Marian DiFiglia

HTT1a was identified in human and mouse Huntington’s disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction. ¹ Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with fluorescence-based reporter assays (Meso Scale Discovery, Homogeneous Time Resolved Fluorescence) and immunoprecipitation assays in Huntington’s disease knock-in mice but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntington’s disease brain were separated by gel electrophoresis and probed by western blot with neo-epitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6 month old Huntington’s disease knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTT1a migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in WT mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating Q111 mice with siRNA to MSH3 , a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear indicating that the effects of curbing CAG repeat expansion were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntington’s disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinson’s disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate HTT proteins from either Huntington’s disease mouse or human brain lysates using conditions that pulled down full length HTT with anti-HTT antibody 2B7. Altogether these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility, and subcellular localization in Huntington’s disease mouse brain. Abbreviated Summary: Sapp et al., report that pathogenic exon 1 protein HTT1a is detected in brain of mouse models of Huntington’s disease by direct western blot assay using monoclonal antibodies 11G2 and 1B12. Lowering MSH3 mRNA in the caudate putamen to prevent CAG repeat expansion reduced levels of HTT1a.

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.

Background: Synaptic changes occur early in patients with Huntington’s disease (HD) and in mouse models of HD. An analysis of synaptic changes in HD transgenic sheep (OVT73) is fitting since they have been shown to have some phenotypes. They also have larger brains, longer lifespan, and greater motor and cognitive capacities more aligned with humans, and can provide abundant biofluids for in vivo monitoring of therapeutic interventions. Objective: The objective of this study was to determine if there were differences between 5- and 10-year-old OVT73 and wild-type (WT) sheep in levels of synaptic proteins in brain and in neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma. Methods: Mutant huntingtin (mHTT) and other proteins were measured by western blot assay in synaptosomes prepared from caudate, motor, and piriform cortex in 5-year-old and caudate, putamen, motor; and piriform cortex in 10-year-old WT and OVT73 sheep. Levels of NfL, a biomarker for neuronal damage increased in many neurological disorders including HD, were examined in CSF and plasma samples from 10-year-old WT and OVT73 sheep using the Simoa NfL Advantage kit. Results: Western blot analysis showed mHTT protein expression in synaptosomes from OVT73 sheep was 23% of endogenous sheep HTT levels at both ages. Significant changes were detected in brain levels of PDE10A, SCN4B, DARPP32, calmodulin, SNAP25, PSD95, VGLUT 1, VAMP1, and Na+/K+-ATPase, which depended on age and brain region. There was no difference in NfL levels in CSF and plasma in OVT73 sheep compared to age-matched WT sheep. Conclusions: These results show that synaptic changes occur in brain of 5- and 10-year-old OVT73 sheep, but levels of NfL in biofluids are unaffected. Altogether, the data support a prodromal disease state in OVT73 sheep that involves the caudate, putamen and cortex.

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer’s disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. Graphical abstract pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

The huntingtin gene mutation interferes with neurogenesis in human fetal cortex

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

Sandwich ELISA-based methods use Abs that target the expanded polyglutamine (polyQ) tract to quantify mutant huntingtin (mHTT). Using Meso Scale Discovery (MSD) assay, the mHTT signal detected with MW1 Ab correlated with polyQ length and doubled with a difference of only 7 glutamine residues between equivalent amounts of purified mHTTexon1 proteins. Similar polyQ length-dependent effects on MSD signals were confirmed using endogenous full length mHTT from brains of Huntington’s disease (HD) knock-in (KI) mice. We used this avidity bias to devise a method to assess average CAG repeat instability at the protein level in a mixed population of HTT proteins present in tissues. Signal detected for average polyQ length quantification at the protein level by our method exhibited a strong correlation with average CAG repeat length at the genomic DNA level determined by PCR method in striatal tissue homogenates from Hdh^Q140 KI mice and in human HD postmortem cortex. This work establishes that CAG repeat instability in mutant HTT is reflected at the protein level.

Human Molecular Genetics, 2013, 22(6), 1112–1131.

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 HD^Q175/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, HD^Q175/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 HD^Q175/Q7 mice stimulates autophagy and lysosome activity, and lowers mHTT, but may also increase a neuronal stress response.

A) Western blot for HTT detected with MAB2166 antibody and B) S830 antibody in crude lysates of the striatum of non-injected HDQ175/Q7 mice at 9 months of age and mice injected with AAV hSyn1 GFP or AAV hSyn1 NUB1. Note full-length wt and mutant HTT