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.

Levels of Synaptic Proteins in Brain and Neurofilament Light Chain in Cerebrospinal Fluid and Plasma of OVT73 Huntington's Disease Sheep Support a Prodromal Disease State.

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.

Age-Dependent Increase in Tau Phosphorylation at Serine 396 in Huntington's Disease Prefrontal Cortex.

BACKGROUND: To date, it is still controversial whether tau phosphorylation plays a role in Huntington's disease (HD), as previous studies demonstrated either no alterations or increases in phosphorylated tau (pTau) in HD postmortem brain and mouse models. OBJECTIVE: The goal of this study was to determine whether total tau and pTau levels are altered in HD. METHODS: Immunohistochemistry, cellular fractionations, and western blots were used to measure total tau and pTau levels in a large cohort of HD and control postmortem prefrontal cortex (PFC). Furthermore, western blots were performed to assess tau, and pTau levels in HD and control isogenic embryonic stem cell (ESC)-derived cortical neurons and neuronal stem cells (NSCs). Similarly, western blots were used to assess tau and pTau levels in HttQ111 and transgenic R6/2 mice. Lastly, total tau levels were assessed in HD and healthy control plasma using Quanterix Simoa assay. RESULTS: Our results revealed that, while there was no difference in total tau or pTau levels in HD PFC compared to controls, the levels of tau phosphorylated at S396 were increased in PFC samples from HD patients 60 years or older at time of death. Additionally, tau and pTau levels were not changed in HD ESC-derived cortical neurons and NSCs. Similarly, total tau or pTau levels were not altered in HttQ111 and transgenic R6/2 mice compared to wild-type littermates. Lastly, tau levels were not changed in plasma from a small cohort of HD patients compared to controls. CONCLUSIONS: Together these findings demonstrate that pTau-S396 levels increase significantly with age in HD PFC.

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.

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~ 32-fold over PS backbone and > 1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~ 6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

Age-dependent increase in tau phosphorylation at serine 396 in Huntington's disease pre-frontal cortex.

Background: To date, it is still controversial whether tau phosphorylation plays a role in Huntington's disease (HD), as previous studies demonstrated either no alterations or increases in phosphorylated tau (pTau) in HD post-mortem brain and mouse models. Objectives: The goal of this study was to determine whether total tau and pTau levels are altered in HD. Methods: Immunohistochemistry, cellular fractionations, and western blots were used to measure tau and pTau levels in a large cohort of HD and control post-mortem prefrontal cortex (PFC). Furthermore, western blots were performed to assess tau, and pTau levels in HD and control isogenic embryonic stem cell (ESC)-derived cortical neurons and neuronal stem cells (NSCs). Similarly, western blots were used to assess tau and pTau in Htt Q111 and transgenic R6/2 mice. Lastly, total tau levels were assessed in HD and healthy control plasma using Quanterix Simoa assay. Results: Our results revealed that, while there was no difference in tau or pTau levels in HD PFC compared to controls, tau phosphorylated at S396 levels were increased in PFC samples from HD patients 60 years or older at time of death. Additionally, tau and pTau levels were not changed in HD ESC-derived cortical neurons and NSCs. Similarly, tau or pTau levels were not altered in Htt Q111 and transgenic R6/2 mice compared to wild-type littermates. Lastly, tau levels were not changed in plasma from a small cohort of HD patients compared to controls. Conclusion: Together these findings demonstrate that pTau-S396 levels increase significantly with age in HD PFC.

Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

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.

A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system.

Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.

Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in Huntington's disease models.

Small interfering RNAs are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS, enabling development of CNS-targeted therapeutics. Many genetically-defined neurodegenerative disorders are dominant, favoring selective silencing of the mutant allele. In some cases, successfully targeting the mutant allele requires targeting single nucleotide polymorphism (SNP) heterozygosities. Here, we use Huntington's disease (HD) as a model. The optimized compound exhibits selective silencing of mutant huntingtin protein in patient-derived cells and throughout the HD mouse brain, demonstrating SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. Targeting a disease-causing allele using RNAi-based therapies could be helpful in a range of dominant CNS disorders where maintaining wild-type expression is essential.

Impaired XK recycling for importing manganese underlies striatal vulnerability in Huntington's disease.

Mutant huntingtin, which causes Huntington's disease (HD), is ubiquitously expressed but induces preferential loss of striatal neurons by unclear mechanisms. Rab11 dysfunction mediates homeostatic disturbance of HD neurons. Here, we report that Rab11 dysfunction also underscores the striatal vulnerability in HD. We profiled the proteome of Rab11-positive endosomes of HD-vulnerable striatal cells to look for protein(s) linking Rab11 dysfunction to striatal vulnerability in HD and found XK, which triggers the selective death of striatal neurons in McLeod syndrome. XK was trafficked together with Rab11 and was diminished on the surface of immortalized HD striatal cells and striatal neurons in HD mouse brains. We found that XK participated in transporting manganese, an essential trace metal depleted in HD brains. Introducing dominantly active Rab11 into HD striatal cells improved XK dynamics and increased manganese accumulation in an XK-dependent manner. Our study suggests that impaired Rab11-based recycling of XK onto cell surfaces for importing manganese is a driver of striatal dysfunction in Huntington's disease.

Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis.

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. 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.

Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities.

Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.

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.

Promise and challenges of dystonia brain banking: establishing a human tissue repository for studies of X-Linked Dystonia-Parkinsonism.

X-Linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease affecting individuals with ancestry to the island of Panay in the Philippines. In recent years there has been considerable progress at elucidating the genetic basis of XDP and candidate disease mechanisms in patient-derived cellular models, but the neural substrates that give rise to XDP in vivo are still poorly understood. Previous studies of limited XDP postmortem brain samples have reported a selective dropout of medium spiny neurons within the striatum, although neuroimaging of XDP patients has detected additional abnormalities in multiple brain regions beyond the basal ganglia. Given the need to fully define the CNS structures that are affected in this disease, we created a brain bank in Panay to serve as a tissue resource for detailed studies of XDP-related neuropathology. Here we describe this platform, from donor recruitment and consent to tissue collection, processing, and storage, that was assembled within a predominantly rural region of the Philippines with limited access to medical and laboratory facilities. Thirty-six brains from XDP individuals have been collected over an initial 4 years period. Tissue quality was assessed based on histologic staining of cortex, RNA integrity scores, detection of neuronal transcripts in situ by fluorescent hybridization chain reaction, and western blotting of neuronal and glial proteins. The results indicate that this pipeline preserves tissue integrity to an extent compatible with a range of morphologic, molecular, and biochemical analyses. Thus the algorithms that we developed for working in rural communities may serve as a guide for establishing similar brain banks for other rare diseases in indigenous populations.

Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling.

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.

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.