Nacc1 Mutation in Mice Models Rare Neurodevelopmental Disorder with Underlying Synaptic Dysfunction.

A missense mutation in the transcription repressor Nucleus accumbens-associated 1 (NACC1) gene at c.892C>T (p.Arg298Trp) on chromosome 19 causes severe neurodevelopmental delay ( Schoch et al., 2017). To model this disorder, we engineered the first mouse model with the homologous mutation (Nacc1+/R284W ) and examined mice from E17.5 to 8 months. Both genders had delayed weight gain, epileptiform discharges and altered power spectral distribution in cortical electroencephalogram, behavioral seizures, and marked hindlimb clasping; females displayed thigmotaxis in an open field. In the cortex, NACC1 long isoform, which harbors the mutation, increased from 3 to 6 months, whereas the short isoform, which is not present in humans and lacks aaR284 in mice, rose steadily from postnatal day (P) 7. Nuclear NACC1 immunoreactivity increased in cortical pyramidal neurons and parvalbumin containing interneurons but not in nuclei of astrocytes or oligodendroglia. Glial fibrillary acidic protein staining in astrocytic processes was diminished. RNA-seq of P14 mutant mice cortex revealed over 1,000 differentially expressed genes (DEGs). Glial transcripts were downregulated and synaptic genes upregulated. Top gene ontology terms from upregulated DEGs relate to postsynapse and ion channel function, while downregulated DEGs enriched for terms relating to metabolic function, mitochondria, and ribosomes. Levels of synaptic proteins were changed, but number and length of synaptic contacts were unaltered at 3 months. Homozygosity worsened some phenotypes including postnatal survival, weight gain delay, and increase in nuclear NACC1. This mouse model simulates a rare form of autism and will be indispensable for assessing pathophysiology and targets for therapeutic intervention.

Early whole-body mutant huntingtin lowering averts changes in proteins and lipids important for synapse function and white matter maintenance in the LacQ140 mouse model.

Expansion of a triplet repeat tract in exon 1 of the HTT gene causes Huntington's disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. Lowering mHTT is a promising approach to treat HD, but it is unclear when lowering should be initiated, how much is necessary, and what duration should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. Using a mHtt-inducible mouse model, we analyzed mHtt lowering initiated at different ages and sustained for different time-periods. mHTT protein in cytoplasmic and synaptic compartments of the striatum was reduced 38-52%; however, there was minimal lowering of mHTT in nuclear and perinuclear regions where aggregates formed at 12 months of age. Total striatal lipids were reduced in 9-month-old LacQ140 mice and preserved by mHtt lowering. Subclasses important for white matter structure and function including ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (MGDG), contributed to the reduction in total lipids. Phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA) were also changed in LacQ140 mice. Levels of all subclasses except ceramide were preserved by mHtt lowering. mRNA expression profiling indicated that a transcriptional mechanism contributes to changes in myelin lipids, and some but not all changes can be prevented by mHtt lowering. Our findings suggest that early and sustained reduction in mHtt can 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.

Early whole-body mutant huntingtin lowering averts changes in proteins and lipids important for synapse function and white matter maintenance in the LacQ140 mouse model.

Expansion of a triplet repeat tract in exon1 of the HTT gene causes Huntington's disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. No disease modifying treatments exist but lowering mutant huntingtin (mHTT) by gene therapy is a promising approach to treat Huntington's disease (HD). It is not clear when lowering should be initiated, how much lowering is necessary and for what duration lowering should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. Using a mHtt-inducible mouse model we analyzed whole body mHtt lowering initiated at different ages and sustained for different time-periods. Subcellular fractionation (density gradient ultracentrifugation), protein chemistry (gel filtration, western blot, and capillary electrophoresis immunoassay), liquid chromatography and mass spectrometry of lipids, and bioinformatic approaches were used to test effects of mHTT transcriptional lowering. 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. mHtt transcript 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 mHtt lowering. The reduction in total lipids was due in part to reductions in subclasses of ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (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 mHtt lowering. Pathway enrichment analysis of RNAseq data imply a transcriptional mechanism is responsible in part for changes in myelin lipids, and some but not all changes can be rescued by mHTT lowering. Our findings suggest that early and sustained reduction in mHtt can 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.

Taming the Huntington's Disease Proteome: What Have We Learned?

Mass spectrometry (MS) is a physical technique used to identify specific chemicals and molecules by precise analysis of their mass and charge; this technology has been adapted for biological sciences applications. Investigators have used MS to identify differential expressions of proteins in Huntington's disease (HD), to discover Huntingtin (HTT) interacting proteins and to analyze HTT proteoforms. Using systems biology and computational approaches, data from MS screens have been leveraged to find differentially expressed pathways. This review summarizes the data from most of the MS studies done in the HD field in the last 20 years and compares it to the protein data reported before the use of MS technology. The MS results validate early findings in the field such as differential expression of PDE10a and DARPP-32 and identify new changes. We offer a perspective on the MS approach in HD, particularly for identification of disease pathways, the challenges in interpreting data across different studies, and its application to protein studies moving forward.

Disposition of Proteins and Lipids in Synaptic Membrane Compartments Is Altered in Q175/Q7 Huntington's Disease Mouse Striatum.

Dysfunction at synapses is thought to be an early change contributing to cognitive, psychiatric and motor disturbances in Huntington's disease (HD). In neurons, mutant Huntingtin collects in aggregates and distributes to the same sites as wild-type Huntingtin including on membranes and in synapses. In this study, we investigated the biochemical integrity of synapses in HD mouse striatum. We performed subcellular fractionation of striatal tissue from 2 and 6-month old knock-in Q175/Q7 HD and Q7/Q7 mice. Compared to striata of Q7/Q7 mice, proteins including GLUT3, Na+/K+ ATPase, NMDAR 2b, PSD95, and VGLUT1 had altered distribution in Q175/Q7 HD striata of 6-month old mice but not 2-month old mice. These proteins are found on plasma membranes and pre- and postsynaptic membranes supporting hypotheses that functional changes at synapses contribute to cognitive and behavioral symptoms of HD. Lipidomic analysis of mouse fractions indicated that compared to those of wild-type, fractions 1 and 2 of 6 months Q175/Q7 HD had altered levels of two species of PIP2, a phospholipid involved in synaptic signaling, increased levels of cholesterol ester and decreased cardiolipin species. At 2 months, increased levels of species of acylcarnitine, phosphatidic acid and sphingomyelin were measured. EM analysis showed that the contents of fractions 1 and 2 of Q7/Q7 and Q175/Q7 HD striata had a mix of isolated synaptic vesicles, vesicle filled axon terminals singly or in clusters, and ER and endosome-like membranes. However, those of Q175/Q7 striata contained significantly fewer and larger clumps of particles compared to those of Q7/Q7. Human HD postmortem putamen showed differences from control putamen in subcellular distribution of two proteins (Calnexin and GLUT3). Our biochemical, lipidomic and EM analysis show that the presence of the HD mutation conferred age dependent disruption of localization of synaptic proteins and lipids important for synaptic function. Our data demonstrate concrete biochemical changes suggesting altered integrity of synaptic compartments in HD mice that may mirror changes in HD patients and presage cognitive and psychiatric changes that occur in premanifest HD.

Protein changes in synaptosomes of Huntington's disease knock-in mice are dependent on age and brain region.

Molecular changes at synapses are thought to underly the deficits in motor and cognitive dysfunction seen in Huntington's disease (HD). Previously we showed in synaptosome preparations age dependent changes in levels of selected proteins examined by western blot assay in the striatum of Q140/Q140 HD mice. To assess if CAG repeat length influenced protein changes at the synapse, we examined synaptosomes from 6-month old heterozygote HD mice with CAG repeat lengths ranging from 50 to 175. Analysis of 19 selected proteins showed that increasing CAG repeat length in huntingtin (HTT) increased the number of affected proteins in HD striatal synaptosomes. Moreover, SDS-soluble total HTT (WT plus mutant HTT) and pThr3 HTT were reduced with increasing CAG repeat length, and there was no pSer421 mutant HTT detected in any HD mice. A LC-MS/MS and bioinfomatics study of synaptosomes from 2 and 6-month old striatum and cortex of Q140/Q7 HD mice showed enrichment of synaptic proteins and an influence of age, gender and brain region on the number of protein changes. HD striatum at 6 months had the most protein changes that included many HTT protein interactors, followed by 2-month old HD striatum, 2-month old HD cortex and 6-month HD cortex. SDS-insoluble mutant HTT was detected in HD striatal synaptosomes consistent with the presence of aggregates. Proteins changed in cortex differed from those in striatum. Pathways affected in HD striatal synaptosomes that were not identified in whole striatal lysates of the same HD mouse model included axon guidance, focal adhesion, neurotrophin signaling, regulation of actin cytoskeleton, endocytosis, and synaptic vesicle cycle. Results suggest that synaptosomes prepared from HD mice are highly informative for monitoring protein changes at the synapse and may be preferred for assessing the effects of experimental therapies on synaptic function in HD.