Comparative Analysis of Acid Sphingomyelinase Distribution in the CNS of Rats and Mice Following Intracerebroventricular Delivery

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we reported that biochemical and functional abnormalities observed in ASM knockout (ASMKO) mice could be partially alleviated by intracerebroventricular (ICV) infusion of hASM. We now show that this route of delivery also results in widespread enzyme distribution throughout the rat brain and spinal cord. However, enzyme diffusion into CNS parenchyma did not occur in a linear dose-dependent fashion. Moreover, although the levels of hASM detected in the rat CNS were determined to be within the range shown to be therapeutic in ASMKO mice, the absolute amounts represented less than 1% of the total dose administered. Finally, our results also showed that similar levels of enzyme distribution are achieved across rodent species when the dose is normalized to CNS weight as opposed to whole body weight. Collectively, these data suggest that the efficacy observed following ICV delivery of hASM in ASMKO mice could be scaled to CNS of the rat

Percentage of the total administered dose of hASM detected in the (A) brain, (B) spinal cord and (C) serum of rats following 6 h ICV infusion.

<p>Animals were infused with 0.1, 1.0 or 10 mg/kg of hASM or aCSF. For both the brain and spinal cord, less than 0.2% of the total dose could be detected in each region. Cumulatively, the level of hASM detected in the brain and spinal cord was less than 1% of the total dose administered. No significant differences were observed between regions or different dose groups. Significant (p<.01) levels of hASM were detected in serum for each dose.</p

hASM levels in (A) rat brain, (B) spinal cord and (C) serum following a 6 h ICV infusion of hASM at different doses (0.1, 1.0 and 10 mg/kg) or aCSF.

<p>Significant (p<.01) levels of hASM were detected in all regions of the brain (i.e., cortex, preoptic region, striatum, hippocampus, thalamus, hypothalamus, midbrain, cerebellum and brainstem) in rats infused with 1 and 10 mg/kg of the enzyme. Rats infused with the 0.1 mg/kg dose displayed significant hASM levels in the cortex, preoptic region, striatum and hippocampus. No significant differences in hASM levels between regions were observed regardless of the dose tested. In each region of the spinal cord, significant (p<.01) levels of hASM were also detected in rats infused at either 1 or 10 mg/kg vs. rats infused with aCSF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016313#pone-0016313-g002" target="_blank">Figure 2b</a>). Detected hASM levels in rats infused with 0.1 mg/kg were not significantly above background. Significant (p<.01) levels of hASM were detected in serum for all hASM infused rats regardless of the dose of enzyme used.</p

(A) Distribution of hASM in the rat CNS following a 6 h ICV infusion of hASM or aCSF (S1 = most rostral brain section and S5 = most caudal brain section, S6 = cervical spinal cord).

<p>Positive hASM staining was observed throughout the brain and spinal cord of rats administered with the enzyme. Staining intensity was maximal in regions proximal to ventricular compartments and the sub-arachnoid space. (B) Higher magnification (4× for brain; 20× for spinal cord) of hASM staining in regions between ventricular compartments and the tissue proximal to the sub-arachnoid space (area enclosed within the white box of sections S2 and S6). Shown are images from the brain and cervical spinal cord.</p

AAV4-mediated Expression of IGF-1 and VEGF Within Cellular Components of the Ventricular System Improves Survival Outcome in Familial ALS Mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron cell death in the cortex, brainstem, and spinal cord. Extensive efforts have been made to develop trophic factor-based therapies to enhance motor neuron survival; however, achievement of adequate therapeutic delivery to all regions of the corticospinal tract has remained a significant challenge. Here, we show that adeno-associated virus serotype 4 (AAV4)-mediated expression of insulin-like growth factor-1 (IGF-1) or vascular endothelial growth factor (VEGF)-165 in the cellular components of the ventricular system including the ependymal cell layer, choroid plexus [the primary cerebrospinal fluid (CSF)-producing cells of the central nervous system (CNS)] and spinal cord central canal leads to trophic factor delivery throughout the CNS, delayed motor decline and a significant extension of survival in SOD1G93A transgenic mice. Interestingly, when IGF-1- and VEGF-165-expressing AAV4 vectors were given in combination, no additional benefit in efficacy was observed suggesting that these trophic factors are acting on similar signaling pathways to modestly slow disease progression. Consistent with these findings, experiments conducted in a recently described in vitro cell culture model of ALS led to a similar result, with both IGF-1 and VEGF-165 providing significant motor neuron protection but in a nonadditive fashion. These findings support the continued investigation of trophic factor-based therapies that target the CNS as a potential treatment of ALS