Prenatal delivery of a therapeutic antisense oligonucleotide achieves broad biodistribution in the brain and ameliorates Angelman syndrome phenotype in mice.

Angelman syndrome (AS), an early-onset neurodevelopmental disorder characterized by abnormal gait, intellectual disabilities, and seizures, occurs when the maternal allele of the UBE3A gene is disrupted, since the paternal allele is silenced in neurons by the UBE3A antisense (UBE3A-AS) transcript. Given the importance of early treatment, we hypothesized that prenatal delivery of an antisense oligonucleotide (ASO) would downregulate the murine Ube3a-AS, resulting in increased UBE3A protein and functional rescue. Using a mouse model with a Ube3a-YFP allele that reports on-target ASO activity, we found that in utero, intracranial (IC) injection of the ASO resulted in dose-dependent activation of paternal Ube3a, with broad biodistribution. Accordingly, in utero injection of the ASO in a mouse model of AS also resulted in successful restoration of UBE3A and phenotypic improvements in treated mice on the accelerating rotarod and fear conditioning. Strikingly, even intra-amniotic (IA) injection resulted in systemic biodistribution and high levels of UBE3A reactivation throughout the brain. These findings offer a novel strategy for early treatment of AS using an ASO, with two potential routes of administration in the prenatal window. Beyond AS, successful delivery of a therapeutic ASO into neurons has implications for a clinically feasible prenatal treatment for numerous neurodevelopmental disorders.

Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections.

In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain. Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3'-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples of procedure results, please see images published in Lopez et al. (2012).

Genetic dissection of a cell-autonomous neurodegenerative disorder: lessons learned from mouse models of Niemann-Pick disease type C.

Understanding neurodegenerative disease progression and its treatment requires the systematic characterization and manipulation of relevant cell types and molecular pathways. The neurodegenerative lysosomal storage disorder Niemann-Pick disease type C (NPC) is highly amenable to genetic approaches that allow exploration of the disease biology at the organismal, cellular and molecular level. Although NPC is a rare disease, genetic analysis of the associated neuropathology promises to provide insight into the logic of disease neural circuitry, selective neuron vulnerability and neural-glial interactions. The ability to control the disorder cell-autonomously and in naturally occurring spontaneous animal models that recapitulate many aspects of the human disease allows for an unparalleled dissection of the disease neurobiology in vivo. Here, we review progress in mouse-model-based studies of NPC disease, specifically focusing on the subtype that is caused by a deficiency in NPC1, a sterol-binding late endosomal membrane protein involved in lipid trafficking. We also discuss recent findings and future directions in NPC disease research that are pertinent to understanding the cellular and molecular mechanisms underlying neurodegeneration in general.

Complement is dispensable for neurodegeneration in Niemann-Pick disease type C.

BACKGROUND: The immune system has been implicated in neurodegeneration during development and disease. In various studies, the absence of complement (that is, C1q deficiency) impeded the elimination of apoptotic neurons, allowing survival. In the genetic lysosomal storage disease Niemann-Pick C (NPC), caused by loss of NPC1 function, the expression of complement system components, C1q especially, is elevated in degenerating brain regions of Npc1-/- mice. Here we test whether complement is mediating neurodegeneration in NPC disease. FINDINGS: In normal mature mice, C1q mRNA was found in neurons, particularly cerebellar Purkinje neurons (PNs). In Npc1-/- mice, C1q mRNA was additionally found in activated microglia, which accumulate during disease progression and PN loss. Interestingly, C1q was not enriched on or near degenerating neurons. Instead, C1q was concentrated in other brain regions, where it partially co-localized with a potential C1q inhibitor, chondroitin sulfate proteoglycan (CSPG). Genetic deletion of C1q, or of the downstream complement pathway component C3, did not significantly alter patterned neuron loss or disease progression. Deletion of other immune response factors, a Toll-like receptor, a matrix metalloprotease, or the apoptosis facilitator BIM, also failed to alter neuron loss. CONCLUSION: We conclude that complement is not involved in the death and clearance of neurons in NPC disease. This study supports a view of neuroinflammation as a secondary response with non-causal relationship to neuron injury in the disease. This disease model may prove useful for understanding the conditions in which complement and immunity do contribute to neurodegeneration in other disorders.

Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C.

Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.

Diastereoselective preparation of (R)- and (S)-2-methoxy-2-phenylpent-3-ynoic acids and their use as reliable chiral derivatizing agents.

Benzoyl-S,O-acetals 1a and 1b were used as chiral auxiliaries to achieve the diastereoselective preparation of both enantiomers of 2-methoxy-2-phenylpent-3-ynoic acids (MPPAs). The latter were condensed with several chiral secondary alcohols and some primary amines to evaluate their potential as chiral derivatizing agents (CDAs). The (1)H NMR spectra of the corresponding esters and amides showed strong consistency with the absolute configuration of the carbinol and amine moieties, whose observed ΔδL(1) and ΔδL(2) values were in the ranges of 0.1-0.4 and 0.02-0.12 ppm, respectively.

A Quick, No Frills Approach to Mouse Genotyping.

Mice are extremely powerful mammalian genetic model organisms for basic and medical research, but managing a colony of transgenic mice is time consuming and expensive, many times requiring the help of dedicated technicians. Slow and laborious genotyping procedures add to the hassle. Outsourcing is costly and may not be as fast as desired, especially when setting up time sensitive experiments. Ultrafast genotyping protocols often require real-time PCR instruments and commercial reagents that may not be economical or practical. This protocol, adapted from methods suggested by The Jackson Laboratory, employs a minimalist approach that maximizes convenience by simplifying the tissue digestion/DNA extraction process and using a high-speed electrophoresis system for sample analysis. Genotype PCR results can be obtained in 3 h or less for as many samples as can fit in a PCR machine or can be efficiently handled by a user. Subsequent ethanol or chloroform purified DNA can be used in a standard PCR reaction to roughly identify a homozygous and a hemizygous mouse.

Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder.

Niemann-Pick type C disease is a fatal lysosomal storage disorder caused by loss of NPC1 function. The disorder severely affects multiple body systems, particularly the nervous system. To test whether rescue of NPC1 activity in neurons, astrocytes, or other cell types can correct the neurological defects, a Tet-inducible Npc1-YFP transgene was introduced into Npc1(-/-) mice for the cell type-specific rescue of NPC1 loss. NPC1-YFP produced in neurons prevented neuron degeneration, slowed reactive glial activity, and ameliorated the disease. NPC1-YFP produced in astrocytes or in cells of visceral tissue did not. These results suggest that loss of NPC1 activity from neurons is the primary cause of the neuropathology and that rescue of NPC1 function in neurons is sufficient to mitigate the disease. The ability of neurons to survive and function in a cell-autonomous fashion allowed the use of this newly engineered rescue system to further define the brain regions or neuron populations required to ameliorate a neurological symptom. NPC1-YFP produced specifically in cerebellar Purkinje neurons reduced ataxia, increased weight, and prolonged life, but it did not prevent the eventual decline and premature death of Npc1(-/-) mice. Significant increase in lifespan correlated with sustained reduction of inflammation in the thalamus. Neuron rescue of other forebrain areas provided little benefit. Future work targeting increasingly discrete neuronal networks should reveal which CNS areas are critical for survival. This work may have broad implications for understanding the anatomical and cellular basis of neurological signs and symptoms of other neurodegenerative and lysosomal disorders.