Effects of Exogenous NUB1 Expression in the Striatum of HDQ175/Q7 Mice.

BACKGROUND: Reducing mutant huntingtin (mHTT) in neurons may be a therapy for Huntington's disease (HD). Elevating NUB1 protein reduced mHTT levels in cell and fly models of HD through a proteasome dependent mechanism. OBJECTIVE: To examine the effects of augmenting NUB1 in HD mouse striatum on mHTT levels. METHODS: Striata of HDQ175/Q7 mice were injected at 3 months of age with recombinant AAV2/9 coding for NUB1 or GFP under the control of the neuron specific human synapsin 1 promoter and examined 6 months post-injection for levels of huntingtin, the striatal markers DARPP32 and PDE10A, the astrocyte marker GFAP, and the autophagy and mHTT aggregate marker P62 using immunolabeling of brain sections and Western blot assay of striatal subcellular fractions. RESULTS: By Western blot human HD brain had only one of the two variants of NUB1 present in human control brain. In striatum of WT and HD mice NUB1 was localized in medium size neurons and enriched in the nucleus of large neurons. In the striatum of NUB1 injected HD mice, there was widespread neuronal distribution of exogenous NUB1 labeling and protein levels were ∼2.5-fold endogenous levels. DARPP32 and GFAP distribution and levels were unchanged but PDE10A levels were lower in crude homogenates and P62 was increased in nuclear enriched P1 fractions. Elevating NUB1 did not change levels of full-length mHTT or the number and size of mHTT (S830) positive nuclear inclusions. CONCLUSION: Findings suggest that increasing NUB1 protein in striatal neurons of HDQ175/Q7 mice in vivo may be relatively safe but is ineffective in reducing mHTT. Increased NUB1 expression in HD striatum alters PDE10A and P62 which are known to be influenced by mHTT.

Autophagy Activation by Transcription Factor EB (TFEB) in Striatum of HDQ175/Q7 Mice.

BACKGROUND: Mutant huntingtin (mHTT) is encoded by the Huntington's disease (HD) gene and its accumulation in the brain contributes to HD pathogenesis. Reducing mHTT levels through activation of the autophagosome-lysosomal pathway may have therapeutic benefit. Transcription factor EB (TFEB) regulates lysosome biogenesis and autophagy. OBJECTIVE: To examine if increasing TFEB protein levels in HD mouse striatum induces autophagy and influences mHTT levels. METHODS: We introduced cDNA encoding TFEB with an HA tag (TFEB-HA) under the control of neuron specific synapsin 1 promoter into the striatum of 3 month old HDQ175/Q7 mice using adeno-associated virus AAV2/9. The levels of exogenous TFEB were analyzed using qPCR and Western blot. Proteins involved in autophagy, levels of huntingtin, and striatal-enriched proteins were examined using biochemical and/or immunohistochemical methods. RESULTS: In HD mice expressing TFEB-HA, HA immunoreactivity distributed throughout the striatum in neuronal cell bodies and processes and preferentially in neuronal nuclei and overlapped with a loss of DARPP32 immunoreactivity. TFEB-HA mRNA and protein were detected in striatal lysates. There were increased levels of proteins involved with autophagosome/lysosome activity including LAMP-2A, LC3II, and cathepsin D and reduced levels of mutant HTT and the striatal enriched proteins DARPP32 and PDE10A. Compared to WT mice, HDQ175/Q7 mice had elevated levels of the ER stress protein GRP78/BiP and with TFEB-HA expression, increased levels of the astrocyte marker GFAP and pro-caspase 3. CONCLUSION: These results suggest that TFEB expression in the striatum of HDQ175/Q7 mice stimulates autophagy and lysosome activity, and lowers mHTT, but may also increase a neuronal stress response.