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Lowering mutant huntingtin (mHTT) transcription is a promising approach to treat Huntington’s disease (HD). Using a mHtt-inducible mouse model we analyzed mHttlowering initiated at different ages and sustained for different time-periods. mHTT protein in cytoplasmic and synaptic compartments of the caudate putamen, which is most affected in HD, was reduced 38-52%. Little or no lowering of mHTT occurred in nuclear and perinuclear regions where aggregates formed at 12 months of age. mHtttranscript repression partially or fully preserved select striatal proteins (SCN4B, PDE10A). Total lipids in striatum were reduced in LacQ140 mice at 9 months and preserved by early partial mHttlowering. The reduction in total lipids was due in part to reductions in subclasses of ceramide (Cer), sphingomyelin (SM), and monogalactosyl diglyceride (MGDG), which are known to be important for white matter structure and function. Lipid subclasses—phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA)—were also changed in LacQ140 mice. Levels of all subclasses other than ceramide were preserved by early mHttlowering. Our findings suggest that early and sustained reduction in mHttcan prevent changes in levels of select striatal proteins and most lipids but a misfolded, degradation-resistant form of mHTT hampers some benefits in the long term.

Small interfering RNAs (siRNAs) are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expressionin vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS. Many genetically-defined neurodegenerative disorders are autosomal dominant favoring selective silencing of the mutant allele. In some cases, successful targeting of the mutant allele requires targeting of a single nucleotide polymorphism (SNP) heterozygosity. Using Huntington’s disease as a model, we demonstrate allele-specific RNAi-based silencing of gene expressionin vivoand in neurons differentiated from HD patient-derived iPSCs. A series ofin vitroscreens, with chemical and thermodynamic optimization, identified compounds with >50-fold selectivity for the mutant HD-causing allele, based on a single nucleotide difference. The optimized compound exhibits selective silencing of mutant huntingtin (HTT) protein in patient derived cells and throughout the HD mouse brain, providing a demonstration of SNP-based allele-specific RNAi silencing of gene expressionin vivoin the CNS. The ability to target a disease-causing allele using RNAi-based therapies could be applied to a wide range of dominant CNS disorders, where maintenance of wild-type expression is essential.

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activation of alternative clearance pathways including Golgi membrane-associated and nucleophagy-based LaminB1 degradation and excretion. The combination of these alternative pathways and canonical autophagy blockade, results in dramatic nuclear pathology with disruption of the nuclear organization, bringing about terminal cell atrophy and degeneration. Thus, our findings identify a novel progressive mechanism for the terminal phases of neuronal cell degeneration and death in human neurodegenerative diseases and provide a link between autophagy block, activation of alternative pathways for degradation, and excretion of cellular components.