Differential effect on myelination through abolition of activity-dependent synaptic vesicle release or reduction of overall electrical activity of selected cortical projections in the mouse.

Myelination of axons by oligodendrocytes in the central nervous system is crucial for fast, saltatory conduction of action potentials. As myelination is central for brain development and plasticity, and deficits are implicated in several neural disorders such as multiple sclerosis, major depressive disorder, bipolar disorder and schizophrenia, it is important to elucidate the underlying mechanisms regulating myelination. Numerous mechanisms have been proposed by which the communication between oligodendrocytes and active axons may regulate the onset and maintenance of activity-dependent myelination. We compared two models of 'silencing' layer V and/or VI cortical projection neurons from early stages by either decreasing their excitability through Kir2.1 expression, an inward rectifying potassium channel, introduced through in utero electroporation at embryonic day (E)13.5, or inhibiting regulated vesicular release through Cre-dependent knock-out of synaptosomal associated protein 25 kDA (SNAP25). SNAP25 is a component of the soluble N-ethylmaleimide fusion protein attachment protein receptor (SNARE) complex, which, among others, is needed for calcium-dependent regulated vesicle release from synapses. In layer VI cortical projection neurons in the Ntsr1-Cre;Ai14;Snap25 fl/fl mouse, we found that inhibiting regulated vesicular release significantly decreased the amount of myelin basic protein (MBP, used as marker for myelination) and the amount of myelinated projections at postnatal day (P)14 without affecting the initial timing of onset of myelination in the brain (at P7/P8). Additionally, overall oligodendrocyte maturation appears to be affected. A strong trend towards reduced node of Ranvier (NoR) length was also observed in Ntsr1-Cre;Ai14;Snap25 fl/fl corpus callosum. An equally strong trend towards reduced NoR length was observed in Rbp4-Cre;Ai14;Snap25 fl/fl corpus callosum at P14, and the g-ratio in the spinal cord dorsal column was reduced at P18. However, no measurable differences in levels of MBP were detected in the striatum when comparing Rbp4-Cre;Ai14;Snap25 fl/fl and control brains. Conversely, Kir2.1 in utero electroporation at E13.5 did not significantly affect the amount of MBP or number of myelinated callosal axons at P14 but did significantly decrease the NoR length measured in the corpus callosum. It therefore seems likely that the excitability of the neuron can potentially perform a modulating function of myelin characteristics, whereas regulated vesicular release has the potential to have a more pronounced effect on overall myelination, but in a cell-type specific manner.

Uncoupling N-acetylaspartate from brain pathology: implications for Canavan disease gene therapy.

N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease-a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.

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

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.