ADAM10 hyperactivation acts on piccolo to deplete synaptic vesicle stores in Huntington's disease.

Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.

RUES2 hESCs exhibit MGE-biased neuronal differentiation and muHTT-dependent defective specification hinting at SP1.

RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.

PD-1 expression is upregulated on adapted T cells in experimental autoimmune encephalomyelitis but is not required to maintain a hyporesponsive state.

T cell adaptation is an important peripheral tolerogenic process which ensures that the T cell population can respond effectively to pathogens but remains tolerant to self-antigens. We probed the mechanisms of T cell adaptation using an experimental autoimmune encephalomyelitis (EAE) model in which the fate of autopathogenic T cells could be followed. We demonstrated that immunisation with a high dose of myelin basic protein (MBP) peptide and complete Freund's adjuvant failed to effectively initiate EAE, in contrast to low dose MBP peptide immunisation which readily induced disease. The proportion of autopathogenic CD4+ T cells in the central nervous system (CNS) of mice immunised with a high dose of MBP peptide was not significantly different to mice immunised with a low dose. However, autopathogenic T cells in mice immunised with high dose MBP peptide had an unresponsive phenotype in ex vivo recall assays. Importantly, whilst expression of PD-1 was increased on adapted CD4+ T cells within the CNS, loss of PD-1 function did not prevent the development of the unresponsive state. The lack of a role for PD-1 in the acquisition of the adapted state stands in striking contrast to the reported functional importance of PD-1 in T cell unresponsiveness in other disease models.

Differentiation of human telencephalic progenitor cells into MSNs by inducible expression of Gsx2 and Ebf1.

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.

Loss of NGF-TrkA signaling from the CNS is not sufficient to induce cognitive impairments in young adult or intermediate-aged mice.

Many molecules expressed in the CNS contribute to cognitive functions either by modulating neuronal activity or by mediating neuronal trophic support and/or connectivity. An ongoing discussion is whether signaling of nerve growth factor (NGF) through its high-affinity receptor TrkA contributes to attention behavior and/or learning and memory, based on its expression in relevant regions of the CNS such as the hippocampus, cerebral cortex, amygdala and basal forebrain. Previous animal models carrying either a null allele or transgenic manipulation of Ngf or Trka have proved difficult in addressing this question. To overcome this problem, we conditionally deleted Ngf or Trka from the CNS. Our findings confirm that NGF-TrkA signaling supports survival of only a small proportion of cholinergic neurons during development; however, this signaling is not required for trophic support or connectivity of the remaining basal forebrain cholinergic neurons. Moreover, comprehensive behavioral analysis of young adult and intermediate-aged mice lacking NGF-TrkA signaling demonstrates that this signaling is dispensable for both attention behavior and various aspects of learning and memory.

hESC-derived striatal progenitors grafted into a Huntington's disease rat model support long-term functional motor recovery by differentiating, self-organizing and connecting into the lesioned striatum.

BACKGROUND: Huntington's disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits. METHODS: With the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function. RESULTS: Human striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution. CONCLUSIONS: The data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.

In vitro-derived medium spiny neurons recapitulate human striatal development and complexity at single-cell resolution.

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD).

Modeling human telencephalic development and autism-associated SHANK3 deficiency using organoids generated from single neural rosettes.

Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.

hiPSCs for predictive modelling of neurodegenerative diseases: dreaming the possible.

Human induced pluripotent stem cells (hiPSCs) were first generated in 2007, but the full translational potential of this valuable tool has yet to be realized. The potential applications of hiPSCs are especially relevant to neurology, as brain cells from patients are rarely available for research. hiPSCs from individuals with neuropsychiatric or neurodegenerative diseases have facilitated biological and multi-omics studies as well as large-scale screening of chemical libraries. However, researchers are struggling to improve the scalability, reproducibility and quality of this descriptive disease modelling. Addressing these limitations will be the first step towards a new era in hiPSC research - that of predictive disease modelling - involving the correlation and integration of in vitro experimental data with longitudinal clinical data. This approach is a key element of the emerging precision medicine paradigm, in which hiPSCs could become a powerful diagnostic and prognostic tool. Here, we consider the steps necessary to achieve predictive modelling of neurodegenerative disease with hiPSCs, using Huntington disease as an example.

The coding and long noncoding single-cell atlas of the developing human fetal striatum.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.

A CRISPR-strategy for the generation of a detectable fluorescent hESC reporter line (WAe009-A-37) for the subpallial determinant GSX2.

GSX2 is a homeobox transcription factor (TF) controlling the specification of the ventral lateral ganglionic eminence and its major derivative, the corpus striatum. Medium spiny neurons (MSNs) represent the largest cell component of the striatum and they are primarily affected in Huntington disease (HD). Here, we used CRISPR technology to generate a pluripotent GSX2-reporter human embryonic stem cell (hESC) line that can be leveraged to monitor striatal differentiation in real-time and to enrich for MSN-committed progenitors.

Stem Cell-Derived Human Striatal Progenitors Innervate Striatal Targets and Alleviate Sensorimotor Deficit in a Rat Model of Huntington Disease.

Huntington disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss and disruption of cortical and basal ganglia circuits. Cell replacement using human embryonic stem cells may offer the opportunity to repair the damaged circuits and significantly ameliorate disease conditions. Here, we showed that in-vitro-differentiated human striatal progenitors undergo maturation and integrate into host circuits upon intra-striatal transplantation in a rat model of HD. By combining graft-specific immunohistochemistry, rabies virus-mediated synaptic tracing, and ex vivo electrophysiology, we showed that grafts can extend projections to the appropriate target structures, including the globus pallidus, the subthalamic nucleus, and the substantia nigra, and receive synaptic contact from both host and graft cells with 6.6 ± 1.6 inputs cell per transplanted neuron. We have also shown that transplants elicited a significant improvement in sensory-motor tasks up to 2 months post-transplant further supporting the therapeutic potential of this approach.

Inhibiting pathologically active ADAM10 rescues synaptic and cognitive decline in Huntington's disease.

A disintegrine and metalloproteinase 10 (ADAM10) is implicated in synaptic function through its interaction with postsynaptic receptors and adhesion molecules. Here, we report that levels of active ADAM10 are increased in Huntington's disease (HD) mouse cortices and striata and in human postmortem caudate. We show that, in the presence of polyglutamine-expanded (polyQ-expanded) huntingtin (HTT), ADAM10 accumulates at the postsynaptic densities (PSDs) and causes excessive cleavage of the synaptic protein N-cadherin (N-CAD). This aberrant phenotype is also detected in neurons from HD patients where it can be reverted by selective silencing of mutant HTT. Consistently, ex vivo delivery of an ADAM10 synthetic inhibitor reduces N-CAD proteolysis and corrects electrophysiological alterations in striatal medium-sized spiny neurons (MSNs) of 2 HD mouse models. Moreover, we show that heterozygous conditional deletion of ADAM10 or delivery of a competitive TAT-Pro-ADAM10709-729 peptide in R6/2 mice prevents N-CAD proteolysis and ameliorates cognitive deficits in the mice. Reduction in synapse loss was also found in R6/2 mice conditionally deleted for ADAM10. Taken together, these results point to a detrimental role of hyperactive ADAM10 at the HD synapse and provide preclinical evidence of the therapeutic potential of ADAM10 inhibition in HD.

LPS-matured CD11c+ bone marrow-derived dendritic cells can initiate autoimmune pathology with minimal injection site inflammation.

The pathogenesis of human autoimmune disorders is incompletely understood. This has led to the development of numerous murine models in which the pathogenesis of autoimmunity can be probed and the efficacy of novel therapies can be tested. One of the most widely-used murine models of autoimmunity is experimental autoimmune encephalomyelitis (EAE). To induce autoimmune pathology, mice are often immunized with an autoantigen alongside an adjuvant, typically complete Freund's adjuvant (CFA). Unfortunately, CFA causes significant inflammation at the site of administration. Despite the well-recognized complication of injection site inflammation, CFA with autoantigen immunization is widely used to induce central nervous system autoimmunity. We performed a literature review which allowed us to estimate that over 10,000 mice were immunized with CFA in published EAE studies in 2013. In this study, we demonstrated that subcutaneously administered myelin basic protein (MBP)-pulsed CD11c+ bone marrow-derived dendritic cells (BMDC) were as effective at inducing EAE as subcutaneously administered MBP plus CFA. Importantly, we also discovered that the CD11c+ BMDC caused significantly less injection site inflammation than MBP plus CFA immunization. This study demonstrated that the use of CD11c+ BMDC can enable the development of autopathogenic T-cells to be studied in vivo without the unwanted side-effects of long-lasting injection site inflammation. This model represents a significant refinement to existing EAE models and may lead to the improvement of the welfare of experimental mice used to study the development of autoimmunity in vivo.

Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA.

Unmethylated CpG-oligodeoxynucleotides (CpG-ODNs) are recognized as a 'danger signal' and are potent immunostimulators. To test whether tumors might be prevented by maintaining the innate immune system on continuous alert, proto-neu transgenic female mice, which develop spontaneous mammary tumors, were systemically treated with CpG-ODNs at 10-day intervals. Tumor incidence and number of tumors/mouse were significantly lower in treated mice compared with the control group. Moreover, CpG-ODN systemic treatment significantly reduced lung metastases induced by intravenous inoculation of N202.1A cells derived from a spontaneous mammary carcinoma. Growth of established tumors was modestly inhibited after CpG-ODN systemic treatment but strongly on peritumoral application. Our data indicate that systemic repeated injection of CpG-ODN to maintain the innate immune system on continuous alert prevents the onset of genetically determined tumors and confers tumor protection when the tumor load is low.

1,25-Dihydroxyvitamin D3-Conditioned CD11c+ Dendritic Cells are Effective Initiators of CNS Autoimmune Disease.

Dendritic cells (DC) play a crucial role in regulating T cell activation. Due to their capacity to shape the immune response, tolerogenic DC have been used to treat autoimmune diseases. In this study, we examined whether 1,25 dihydroxyvitamin D3-conditioned bone marrow-derived DC (VitD-BMDC) were able to limit the development of autoimmune pathology in experimental autoimmune encephalomyelitis (EAE). We found that VitD-BMDC had lower expression of MHC class II and co-stimulatory molecules and were less effective at priming autoreactive T cells in vitro. Using our recently described BMDC-driven model of EAE, we demonstrated that VitD-BMDC had a significantly reduced ability to initiate EAE. We found that the impaired ability of VitD-BMDC to initiate EAE was not due to T cell tolerization. Instead, we discovered that the addition of 1,25(OH)2D3 to BMDC cultures resulted in a significant reduction in the proportion of CD11c+ cells. Purified CD11c+ VitD-BMDC were significantly less effective at priming T cells in vitro yet were similarly capable of initiating EAE as vehicle-treated CD11c+ BMDC. This study demonstrates that in vitro assays of DC function can be a poor predictor of in vivo behavior and that CD11c+ VitD-BMDC are highly effective initiators of an autopathogenic T cell response.

BDNF-TrkB signaling in striatopallidal neurons controls inhibition of locomotor behavior.

The physiology of brain-derived neurotrophic factor signaling in enkephalinergic striatopallidal neurons is poorly understood. Changes in cortical Bdnf expression levels, and/or impairment in brain-derived neurotrophic factor anterograde transport induced by mutant huntingtin (mHdh) are believed to cause striatopallidal neuron vulnerability in early-stage Huntington's disease. Although several studies have confirmed a link between altered cortical brain-derived neurotrophic factor signaling and striatal vulnerability, it is not known whether the effects are mediated via the brain-derived neurotrophic factor receptor TrkB, and whether they are direct or indirect. Using a novel genetic mouse model, here, we show that selective removal of brain-derived neurotrophic factor-TrkB signaling from enkephalinergic striatal targets unexpectedly leads to spontaneous and drug-induced hyperlocomotion. This is associated with dopamine D2 receptor-dependent increased striatal protein kinase C and MAP kinase activation, resulting in altered intrinsic activation of striatal enkephalinergic neurons. Therefore, brain-derived neurotrophic factor/TrkB signaling in striatopallidal neurons controls inhibition of locomotor behavior by modulating neuronal activity in response to excitatory input through the protein kinase C/MAP kinase pathway.

Targeting Plk1 to chromosome arms and regulating chromosome compaction by the PICH ATPase.

During mitosis, chromosomes undergo dynamic structural changes that include condensation of chromosomes-the formation of individual compact chromosomes necessary for faithful segregation of sister chromatids in anaphase. Polo-like kinase 1 (Plk1) regulates multiple mitotic events by binding to targeting factors at different mitotic structures in a phosphorylation dependent manner. In this study, we report the identification of a putative ATPase that targets Plk1 to chromosome arms during mitosis. PICH (Plk1-interacting checkpoint "helicase") displays a temporal localization on chromosome arms and kinetochores during early mitosis. Interaction with PICH recruits Plk1 to chromosome arms and disruption of this interaction abolishes Plk1 localization on chromosome arms. Moreover, depletion of PICH or overexpression of PICH mutant that is defective in Plk1 binding or ATP binding causes defects in mitotic chromosome compaction, formation of anaphase bridge and cytokinesis failure. We provide data to show that both PICH phosphorylation and its ATPase activity are required for mitotic chromosome compaction. Our study provides a mechanism for targeting Plk1 to chromosome arms and suggests that the PICH ATPase activity is important for the regulation of mitotic chromosome architecture.

A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints.

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

Antitumor activity of the TLR-5 ligand flagellin in mouse models of cancer.

Flagellin, the structural protein subunit of the bacterial flagellum, is specifically recognized by TLR-5 and has potent immunomodulatory effects. The antitumor effects of purified Salmonella typhimurium flagellin were evaluated in mice transplanted s.c. with a weakly immunogenic murine tumor or with its variant stably transfected to express the highly antigenic human HER-2 oncoprotein. Peritumoral administration of flagellin 8-10 days after tumor implantation did not affect the growth rate of the weakly immunogenic tumor but significantly inhibited growth of the antigenic variant tumor. In contrast, flagellin administered at the time of implantation of the antigenic tumor led to accelerated tumor growth. These contrasting effects of flagellin on tumor growth correlated with the type of immune response induced; i.e., late flagellin administration was associated with an increased IFN-gamma:IL-4 ratio and the decreased frequency of CD4+CD25+ T regulatory cells, whereas flagellin treatment at the time of tumor implantation decreased the IFN-gamma:IL-4 ratio and increased CD4+CD25+ T cell frequency. When the early flagellin treatment was combined with administration of CpG-containing oligodeoxynucleotides, tumor growth was completely suppressed, indicating synergy between flagellin and CpG-containing oligodeoxynucleotides. Together, these data provide evidence that flagellin can have contrasting effects on tumor growth.