Our data suggest that the distinct mononuclear phagocytic cell response seen in cerebral X-ALD results, at least in part, from aberrant signaling to cognate receptors on microglia. Our findings support a hypothesis that microglial apoptosis in perilesional white matter represents an early stage in lesion evolution and may be an appropriate target for intervention in X-ALD patients with evidence of cerebral demyelination.

To circumvent the limitations of using postmortem brain in molecular assays, we used avidin-biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15-20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and ␤-actin mRNA (SPION-cfos and SPION-␤actin, respectively) (14 -22 nm). The Stern-Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 ϫ 10 6 /M. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (

We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) for in vivo magnetic resonance imaging (MRI) of fosB and ⌬fosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and ⌬fosB probes was verified by in vitro reverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPIONlabeled ⌬fosB probe signal elevation occurred only in chronically AMPH-exposed mice. The in vivo target specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and ⌬fosB mRNA in living brains.

X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.