Proteomic Analysis of Huntington's Disease Medium Spiny Neurons Identifies Alterations in Lipid Droplets.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium-spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72-induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis. Using data-dependent acquisitions with FAIMS for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6323 proteins with at least two unique peptides. Of these, 901 proteins were altered significantly more in the HD72-MSNs than in isogenic controls. Functional enrichment analysis of upregulated proteins demonstrated extracellular matrix and DNA signaling (DNA replication pathway, double-strand break repair, G1/S transition) with the highest significance. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A:EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-γ signaling, immune system major histocompatibility complex, lipid metabolism, and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., septin protein members) were dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and using quantitative image analysis, we found that lipid droplets accumulated in the HD72-MSN, suggesting a deficit in the turnover of lipids possibly through lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.

Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates.

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.

Novel probes for label-free detection of neurodegenerative GGGGCC repeats associated with amyotrophic lateral sclerosis.

DNA repeat expansion sequences cause a myriad of neurological diseases when they expand beyond a critical threshold. Previous electrochemical approaches focused on the detection of trinucleotide repeats (CAG, CGG, and GAA) and relied on labeling of the probe and/or target strands or enzyme-linked assays. However, detection of expanded GC-rich sequences is challenging because they are prone to forming secondary structures such as cruciforms and quadruplexes. Here, we present label-free detection of hexanucleotide GGGGCC repeat sequences, which cause the leading genetic form of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The approach relies on capturing targets by surface-bound oligonucleotide probes with a different number of complementary repeats, which proportionately translates the length of the target strands into charge transfer resistance (RCT) signal measured by electrochemical impedance spectroscopy. The probe carrying three tandem repeats transduces the number of repeats into RCT with a 3× higher calibration sensitivity and detection limit. Chronocoulometric measurements show a decrease in surface density with increasing repeat length, which is opposite of the impedance trend. This implies that the length of the target itself can contribute to amplification of the impedance signal independent of the surface density. Moreover, the probe can distinguish between a control and patient sequences while remaining insensitive to non-specific Huntington's disease (CAG) repeats in the presence of a complementary target. This label-free strategy might be applied to detect the length of other neurodegenerative repeat sequences using short probes with a few complementary repeats. Graphical abstract Short oligomeric probes with multiple complementary repeats detect long neurodegenerative targets with high sensitivity and transduce into higher impedance signal.

Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes.

Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins.

Systematic comparative analysis of strand-specific RNA-seq library preparation methods for low input samples.

Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increases the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies-Swift RNA and Swift Rapid RNA, presently offered by Integrated DNA Technologies (IDT) -alongside the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark gene expression in these kits, at input quantities ranging between 10 to 500 ng. We found normalized read counts between all treatment groups to be in high agreement. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits offer shorter workflow times enabled by their patented Adaptase technology. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways directly attributable to input mRNA amount.

Modulating FKBP5/FKBP51 and autophagy lowers HTT (huntingtin) levels.

Current disease-modifying therapies for Huntington disease (HD) focus on lowering mutant HTT (huntingtin; mHTT) levels, and the immunosuppressant drug rapamycin is an intriguing therapeutic for aging and neurological disorders. Rapamycin interacts with FKBP1A/FKBP12 and FKBP5/FKBP51, inhibiting the MTORC1 complex and increasing cellular clearance mechanisms. Whether the levels of FKBP (FK506 binding protein) family members are altered in HD models and if these proteins are potential therapeutic targets for HD have not been investigated. Here, we found levels of FKBP5 are significantly reduced in HD R6/2 and zQ175 mouse models and human HD isogenic neural stem cells and medium spiny neurons derived from induced pluripotent stem cells. Moreover, FKBP5 interacts and colocalizes with HTT in the striatum and cortex of zQ175 mice and controls. Importantly, when we decreased FKBP5 levels or activity by genetic or pharmacological approaches, we observed reduced levels of mHTT in our isogenic human HD stem cell model. Decreasing FKBP5 levels by siRNA or pharmacological inhibition increased LC3-II levels and macroautophagic/autophagic flux, suggesting autophagic cellular clearance mechanisms are responsible for mHTT lowering. Unlike rapamycin, the effect of pharmacological inhibition with SAFit2, an inhibitor of FKBP5, is MTOR independent. Further, in vivo treatment for 2 weeks with SAFit2, results in reduced HTT levels in both HD R6/2 and zQ175 mouse models. Our studies establish FKBP5 as a protein involved in the pathogenesis of HD and identify FKBP5 as a potential therapeutic target for HD.Abbreviations : ACTB/ß-actin: actin beta; AD: Alzheimer disease; BafA1: bafilomycin A1; BCA: bicinchoninic acid; BBB: blood brain barrier; BSA: bovine serum albumin; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FKBPs: FK506 binding proteins; HD: Huntington disease; HTT: huntingtin; iPSC: induced pluripotent stem cells; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MES: 2-ethanesulfonic acid; MOPS: 3-(N-morphorlino)propanesulfonic acid); MSN: medium spiny neurons; mHTT: mutant huntingtin; MTOR: mechanistic target of rapamycin kinase; NSC: neural stem cells; ON: overnight; PD: Parkinson disease; PPIase: peptidyl-prolyl cis/trans-isomerases; polyQ: polyglutamine; PPP1R1B/DARPP-32: protein phosphatase 1 regulatory inhibitor subunit 1B; PTSD: post-traumatic stress disorder; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST:Tris-buffered saline, 0.1% Tween 20; TUBA: tubulin; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: littermate controls.

Progranulin expression is upregulated after spinal contusion in mice.

Progranulin (proepithelin) is a pleiotropic growth-factor associated with inflammation and wound repair in peripheral tissues. It also has been implicated in the response to acute traumatic brain injury as well as to chronic neurodegenerative diseases. To determine whether changes in progranulin expression also accompany acute spinal cord injury, C57BL/6 mice were subjected to mid-thoracic (T9 level) contusion spinal cord injury and analyzed by immunohistochemical and biochemical methods. Whereas spinal cord sections prepared from non-injured laminectomy control animals contained low basal levels of progranulin immunoreactivity in gray matter, sections from injured animals contained intense immunoreactivity throughout the injury epicenter that peaked 7-14 days post injury. Progranulin immunoreactivity colocalized with myeloid cell markers CD11b and CD68, indicating that expression increased primarily in activated microglia and macrophages. Immunoblot analysis confirmed that progranulin protein levels rose after injury. On the basis of quantitative polymerase chain reaction analysis, increased protein levels resulted from a tenfold rise in progranulin transcripts. These data demonstrate that progranulin is dramatically induced in myeloid cells after experimental spinal cord injury and is positioned appropriately both spatially and temporally to influence recovery after injury.

Characterization and application of fluidic properties of trinucleotide repeat sequences by wax-on-plastic microfluidics.

Trinucleotide repeat (TNR) sequences introduce sequence-directed flexibility in the genomic makeup of all living species leading to unique non-canonical structure formation. In humans, the expansions of TNR sequences are responsible for almost 24 neurodegenerative and neuromuscular diseases because their unique structures disrupt cell functions. The biophysical studies of these sequences affect their electrophoretic mobility and spectroscopic signatures. Here, we demonstrate a novel strategy to characterize and discriminate the TNR sequences by monitoring their capillary flow in the absence of an external driving force using wax-on-plastic microchannels. The wax-on-plastic microfluidic system translates the sequence-directed flexibility of TNR into differential flow dynamics. Several variables were used to characterize sequences including concentration, single- vs. double-stranded samples, type of repeat sequence, length of the repeat sequence, presence of mismatches in duplex, and presence of metal ion. All these variables were found to influence the flow velocities of TNR sequences as these factors directly affect the structural flexibility of TNR at the molecular level. An overall trend was observed as the higher flexibility in the TNR structure leads to lower capillary flow. After testing samples derived from relevant cells harboring expanded TNR sequences, it is concluded that this approach may transform into a reagent-free and pump-free biosensing platform to detect microsatellite expansion diseases.

FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16INK4a increase.

Neurodegenerative diseases (ND) have been linked to the critical process in aging-cellular senescence. However, the temporal dynamics of cellular senescence in ND conditions is unresolved. Here, we show senescence features develop in human Huntington's disease (HD) neural stem cells (NSCs) and medium spiny neurons (MSNs), including the increase of p16INK4a , a key inducer of cellular senescence. We found that HD NSCs reprogram the transcriptional targets of FOXO3, a major cell survival factor able to repress cell senescence, antagonizing p16INK4a expression via the FOXO3 repression of the transcriptional modulator ETS2. Additionally, p16INK4a promotes cellular senescence features in human HD NSCs and MSNs. These findings suggest that cellular senescence may develop during neuronal differentiation in HD and that the FOXO3-ETS2-p16INK4a axis may be part of molecular responses aimed at mitigating this phenomenon. Our studies identify neuronal differentiation with accelerated aging of neural progenitors and neurons as an alteration that could be linked to NDs.

Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking.

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

Modeling Polyglutamine Expansion Diseases with Induced Pluripotent Stem Cells.

Polyglutamine expansion disorders, which include Huntington's disease, have expanded CAG repeats that result in polyglutamine expansions in affected proteins. How this specific feature leads to distinct neuropathies in 11 different diseases is a fascinating area of investigation. Most proteins affected by polyglutamine expansions are ubiquitously expressed, yet their mechanisms of selective neurotoxicity are unknown. Induced pluripotent stem cells have emerged as a valuable tool to model diseases, understand molecular mechanisms, and generate relevant human neural and glia subtypes, cocultures, and organoids. Ideally, this tool will generate specific neuronal populations that faithfully recapitulate specific polyglutamine expansion disorder phenotypes and mimic the selective vulnerability of a given disease. Here, we review how induced pluripotent technology is used to understand the effects of the disease-causing polyglutamine protein on cell function, identify new therapeutic targets, and determine how polyglutamine expansion affects human neurodevelopment and disease. We will discuss ongoing challenges and limitations in our use of induced pluripotent stem cells to model polyglutamine expansion diseases.

Nuclear Receptor Nr4a1 Regulates Striatal Striosome Development and Dopamine D1 Receptor Signaling.

The GABAergic medium-size spiny neuron (MSN), the striatal output neuron, may be classified into striosome, also known as patch, and matrix, based on neurochemical differences between the two compartments. At this time, little is known regarding the regulation of the development of the two compartments. Nr4a1, primarily described as a nuclear receptor/immediate early gene involved in the homeostasis of the dopaminergic system, is a striosomal marker. Using Nr4a1-overexpressing and Nr4a1-null mice, we sought to determine whether Nr4a1 is necessary and/or sufficient for striosome development. We report that in vivo and in vitro, Nr4a1 and Oprm1 mRNA levels are correlated. In the absence of Nr4a, there is a decrease in the percentage of striatal surface area occupied by striosomes. Alterations in Nr4a1 expression leads to dysregulation of multiple mRNAs of members of the dopamine receptor D1 signal transduction system. Constitutive overexpression of Nr4a1 decreases both the induction of phosphorylation of ERK after a single cocaine exposure and locomotor sensitization following chronic cocaine exposure. Nr4a1 overexpression increases MSN excitability but reduces MSN long-term potentiation. In the resting state, type 5 adenylyl cyclase (AC5) activity is normal, but the ability of AC5 to be activated by Drd1 G-protein-coupled receptor inputs is decreased. Our results support a role for Nr4a1 in determination of striatal patch/matrix structure and in regulation of dopaminoceptive neuronal function.

Altered Expression of Matrix Metalloproteinases and Their Endogenous Inhibitors in a Human Isogenic Stem Cell Model of Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a progressive movement disorder, psychiatric symptoms, and cognitive impairments. HD is caused by a CAG repeat expansion encoding a stretch of polyglutamine residues in the N-terminus of mutant huntingtin (mHTT) protein. Proteolytic processing of mHTT yields toxic fragments, which cause neurotoxicity and massive neuronal cell death predominantly in the striatum and cortex. Inhibition of mHTT cleavage reduces neuronal toxicity suggesting mHTT proteolysis contributes to HD pathogenesis. A previously conducted unbiased siRNA screen in our lab for known human proteases identified matrix metalloproteinases (MMPs) as modifiers of mHTT proteolysis and toxicity. To further study MMP activation in HD, isogenic HD, and control corrected (C116) neural stem cells (NSCs) prepared from HD patient-derived induced pluripotent stem cells were used to examine the role of MMPs and their endogenous inhibitors in this highly relevant model system. We found altered expression of MMP-2 and MMP-9 (gelatinases), MMP-3/10, and MMP-14, activity in HD-NSCs when compared to control C116-NSCs. Dysregulation in MMP activity was accompanied with concomitant changes in levels of endogenous inhibitors of MMPs, called tissue inhibitors of matrix metalloproteinases (TIMPs). Specifically, we observed decreased levels of TIMP-1 and TIMP-2 in HD-NSCs, suggesting part of the altered expression and activity of MMPs is due to lower abundance of these endogenous inhibitors. Immunofluorescence analysis revealed increased MMP/TIMP localization in the nucleus or aggregates of HD-NSCs, suggesting potential interaction with mHTT. TIMP-1 was found to associate with mHTT aggregates in discrete punctate structures in HD-NSCs. These events collectively contribute to increased neurotoxicity in HD. Previous characterization of these NSCs revealed transforming growth factor beta (TGF-ß) pathway as the top dysregulated pathway in HD. TGF-ß was significantly upregulated in HD-NSCs and addition of TGF-ß to HD-NSCs was found to be neuroprotective. To determine if TGF-ß regulated MMP and TIMP activity, C116- and HD-NSCs were exogenously treated with recombinant TGF-ß. TIMP-1 levels were found to be elevated in response to TGF-ß treatment, representing a potential mechanism through which elevated TGF-ß levels confer neuroprotection in HD. Studying the mechanism of action of MMPs and TIMPs, and their interactions with mHTT in human isogenic patient-derived NSCs elucidates new mechanisms of HD neurotoxicity and will likely provide novel therapeutics for treatment of HD.

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation.

PURPOSE: Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis. METHODS: We isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein. RESULTS: We found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells. CONCLUSIONS: To our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies.

Research towards tau imaging.

Tau-bearing neurofibrillary lesions present a promising biomarker for premortem diagnosis and staging of Alzheimer's disease and certain forms of frontotemporal lobar degeneration by whole brain imaging methods. Although brain penetrating compounds capable of binding tau aggregates with high affinity have been disclosed for this purpose, the major barrier to progress remains the need for tau lesion binding selectivity relative to amyloid-beta plaques and other deposits of proteins in cross-beta-sheet conformation. Here we discuss challenges faced in the development of tau lesion-selective imaging agents, and recent preclinical advances in pursuit of this goal.

Modulation and detection of tau aggregation with small-molecule ligands.

Recent results from high-throughput and other screening approaches reveal that small molecules can directly interact with recombinant full-length tau monomers and fibrillar tau aggregates in three distinct modes. First, in the high concentration regime (>10 micromolar), certain anionic molecules such as Congo red efficiently promote tau filament formation through a nucleation-elongation mechanism involving a dimeric nucleus and monomer-mediated elongation. These compounds are useful for modeling tau aggregation in vitro and in biological models. Second, in the low concentration regime (<1 micromolar), other ligands, including cyanine dyes, display aggregation antagonist activity. Compounds that can prevent or reverse fibrillization are candidate modifiers of disease pathology. Finally, certain compounds bind mature tau fibrils with varying affinities at multiple binding sites without modulating the aggregation reaction. For some ligands, >10-fold selectivity for tau aggregates relative to filaments composed of beta-amyloid or alpha-synuclein can be demonstrated at the level of binding affinity. Together these observations suggest that small-molecules have utility for interrogating the tau aggregation pathway, for inhibiting neuritic lesion formation, and for selective pre-mortem detection of neurofibrillary lesions through whole brain imaging.