Loss of Central Auditory Processing in a Mouse Model of Canavan Disease

<div><p>Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the <i>Aspa^lacZ/lacZ</i> rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from <i>Aspa^lacZ/lacZ</i> mice, indicated altered central auditory processing in mutant mice compared with <i>Aspa^wt/wt</i> controls and altered central auditory processing. Analysis of ABR latencies recorded from <i>Aspa^lacZ/lacZ</i> mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of <i>Aspa^lacZ/lacZ</i> mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.</p></div

ABR recordings reveal hypoacusis in <i>Aspa^lacZ/lacZ</i> mice at 4 months.

<p>Representative ABR waveforms from (A) <i>Aspa^wt/wt</i> and (B) <i>Aspa^lacZ/lacZ</i> mice elicited by click stimulus. Note the substantial reduction of P4 and absence of P5 in the mutant waveform. Arrows indicate ABR thresholds. (C) ABR responses of <i>Aspa^lacZ/lacZ</i> mice have higher thresholds for the click stimulus (29.0±1.0 db, n = 5) compared with <i>Aspa^wt/wt</i> controls (23.0±1.2 dB, n = 5; p = 0.027). ABR hearing threshold was not significantly different at 16 kHz (<i>Aspa^wt/wt</i> 13.0±1.2 dB; <i>Aspa^lacZ/lacZ</i> 18.0±2.5 dB; p = 0.143) or at 24 kHz (<i>Aspa^wt/wt</i> 27.0±3.4 dB; <i>Aspa^lacZ/lacZ</i> 27.0±2.0 dB; p = 0.782). (D) Analyses of peak latencies in response to click stimuli (30 dB above threshold) showed P1 were similar between <i>Aspa^lacZ/lacZ</i> mice (1.62±0.03 ms, n = 5) and <i>Aspa^wt/wt</i> controls (1.59±0.03 ms, n = 5; p = 0.633), yet with significant differences for P2 (<i>Aspa^lacZ/lacZ</i>, 2.49±0.07 ms; <i>Aspa^wt/w</i>, 2.25±0.04 ms; p<0.001) and P3 (<i>Aspa^lacZ/lacZ</i>, 3.40±0.08 ms; <i>Aspa^wt/w</i>, 3.08±0.04 ms; p<0.001). Analysis of data was precluded for P4 and P5 due to the absence of the corresponding features in ABR waveforms from <i>aspa^lacZ/lacZ</i> animals. All data were analyzed by two-way ranked ANOVA and Holm-Sidak post-hoc comparison.</p

Hypomyelination of central auditory structures in ASPA-deficient mice.

<p>Luxol Fast Blue staining of brain sections from <i>Aspa^wt/wt</i> mice (A, C, E, G, I) and <i>Aspa^lacZ/lacZ</i> mice (B, D, F, H, J) reveals demyelination in the brainstem (A–H) and midbrain (I, J) of the mutant. Hypomyelination is severe in white matter of the VIII cranial nerve adjacent to the cochlear nucleus (A, B), or axon tracts of the lateral lemniscus (G, H). Differences were less evident in inherently myelin-poor grey matter of the cochlear nucleus (C, D), superior olivary complex (E, F), and inferior colliculus (I, J). Note that Luxol Fast Blue-treated sections were counterstained with Cresyl Violet. VIII cranial nerve, VIII; cochlear nucleus, CN; superior olivary complex, SOC; lateral lemniscus, LL; inferior colliculus, CIC. Bars: 20 µm.</p

Central histopathology of <i>Aspa^lacZ/lacZ</i> mice.

<p>(A–L) Representative overviews of coronal sections from <i>Aspa^wt/w</i> mice and <i>Aspa^lacZ/lacZ</i> mice, stained with H&E (purple) and Luxol Fast Blue (blue) to visualize gross tissue integrity and myelination, respectively. Sections from forebrain (A–D), midbrain (E–H), and hindbrain (I–L) regions illustrate that vacuolization in <i>Aspa^lacZ/lacZ</i> mice is moderate in the neocortex but prominent in posterior regions including the hippocampus, thalamus, cerebellar white matter, and dorsal brainstem. Widespread demyelination is observed by reduced intensity of the Luxol Fast Blue signal, particularly in white matter. Boxes in (I–L) indicate the brainstem region containing the cochlear nucleus and VIII cranial nerve. (I’–L’) Higher magnification of the areas containing the cochlear nucleus (CN). Arrowheads indicate myelination deficits in the VIII cranial nerve (K’–L’). Bars: A–L, 1 mm; I’–L’, 100 µm.</p

Normal cochlear anatomy and outer hair cell function in <i>Aspa^lacZ/lacZ</i> mice.

<p>Representative transmitted light laser scanning microscopy images of midmodiolar sections of the cochleae of <i>Aspa^wt/wt</i> (A) and <i>Aspa^lacZ/lacZ</i> mice (B) revealed normal gross anatomical organization of the cochlea including preserved organ of Corti (o/C), spiral ganglia (sg), Reissner's membrane (rm), scala vestibuli (SV), scala media (SM), scala tympany (ST), cochlear nerve (cn). (C, D) Close-up of β-III tubulin-expressing spiral ganglia neurons (sgn; red) showed no abnormalities. DAPI (blue) was used to label nuclei. (E, F) High power images of the organ of Corti, with the innervation of the hair cells (neurofilament immunofluorescence, red) overlaid on the transmitted light images. Inner hair cells (ihc) are appropriately innervated by spiral ganglion neurites. DAPI (blue) was used for counterstain. ohc, outer hair cells; Dc, Deiters' cells. (G) 2f₁-f₂ DPOAEs recorded at different primary tone frequencies showed normal thresholds, implying normal OHC electromotility and cochlear amplifier response in <i>Aspa^lacZ/lacZ</i> mice compared with <i>Aspa^wt/wt</i> controls. Two-way ANOVA on ranked data and Holm-Sidak post-hoc comparison analyses showed no genotype differences for 8 kHz (<i>Aspa^lacZ/lacZ</i>, 14.0±1.9; <i>Aspa^wt/w</i>, 13.0±1.2, p = 0.658), 16 kHz (<i>Aspa^lacZ/lacZ</i>, 22.0±2.5; <i>Aspa^wt/w</i>, 22.0±2.5, p = 1.0), or 24 kHz (<i>Aspa^lacZ/lacZ</i>, 40.0±6.1; <i>Aspa^wt/w</i>, 42.0±1.2, p = 0.556). n = 5; Bars: A–B, 100 µm; C–F, 20 µm.</p

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<p>Translation of mRNA into protein is an evolutionarily conserved, fundamental process of life. A prerequisite for translation is the accurate charging of tRNAs with their cognate amino acids, a reaction catalyzed by specific aminoacyl-tRNA synthetases. One of these enzymes is the aspartyl-tRNA synthetase DARS, which pairs aspartate with its corresponding tRNA. Missense mutations of the gene encoding DARS result in the leukodystrophy hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL) with a distinct pattern of hypomyelination, motor abnormalities, and cognitive impairment. A thorough understanding of the DARS expression domains in the central nervous system is essential for the development of targeted therapies to treat HBSL. Here, we analyzed endogenous DARS expression on the mRNA and protein level in different brain regions and cell types of human post mortem brain tissue as well as in human stem cell derived neurons, oligodendrocytes, and astrocytes. DARS expression is significantly enriched in the cerebellum, a region affected in HBSL patients and important for motor control. Although obligatorily expressed in all cells, DARS shows a distinct expression pattern with enrichment in neurons but only low abundance in oligodendrocytes, astrocytes, and microglia. Our results reveal little homogeneity across the different cell types, largely matching previously published data in the murine brain. This human gene expression study will significantly contribute to the understanding of DARS gene function and HBSL pathology and will be instrumental for future development of animal models and targeted therapies. In particular, we anticipate high benefit from a gene replacement approach in neurons of HBSL mouse models, given the abundant endogenous DARS expression in this lineage cell.</p

Presentation_1_Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.pdf

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the ‘cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, “contralateral suppression” of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24–32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.</p

Glial Promoter Selectivity following AAV-Delivery to the Immature Brain

<div><p>Recombinant adeno-associated virus (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP) expression driven by the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA)-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.</p></div

Astrocytic transgene expression after neonatal delivery of AAV-GFAP-GFP.

<p>AAV (2×10⁹ vg) was administered to the striatum of newborn mice. Brains (n = 3) were analyzed three weeks later for GFP expression (green) in combination with cell-type specific markers (red). Immunoreactivities of GFP with ASPA (A), or NeuN (B) segregated. C, Co-staining with ALDH1L1 identified GFP⁺ cells as astrocytes (arrows). D, Quantitative comparison of neural populations expressing GFP shows selectivity of the GFAP promoter in astrocytes. E, Percentage of GFP⁺ cells among individual neural populations in the target region showing transgene expression in 50% of astrocytes. In contrast, only negligible numbers of oligodendrocytes or neurons expressed the transgene. Bar: 50 µm.</p

Promoter selectivity targets transgene expression to specific neural cell types <i>in vitro</i> and in the adult brain.

<p>AAV vectors (1×10⁹ vg) were used to express GFP driven by the indicated promoters in enriched primary oligodendrocyte cultures (A–C). For in vivo studies vectors (2×10⁹ vg) were injected in the striatum of adult mice (D–F). Representative images of results by double-immunocytochemistry for GFP (green) and cell-type specific markers (red) illustrate promoter selectivity. A, In primary cultures AAV-CBA-GFP expressed in all NeuN⁺ neurons. In addition, NeuN-negative astrocytes (top in picture) showed GFP immunoreactivity. B, AAV-MBP-GFP-mediated GFP-expression was restricted to ASPA⁺ oligodendrocytes in vitro. C, AAV-GFAP-GFP transduction resulted in GFP immunoreactivity limited to cultured GFAP⁺ astrocytes. D, CBA promoter-controlled GFP expression was highly specific to neurons in vivo. E, The MBP promoter was selective for forebrain oligodendrocytes. F, The GFAP promoter drove GFP specifically in astrocytes. Representative results from three independent experiments are shown. Bars: A–C, 50 µm; D–F, 100 µm.</p