Targeting thalamic circuits rescues motor and mood deficits in PD mice.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification.

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version-a major advance-based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

Mixed T Cell Chimerism After Allogeneic Hematopoietic Stem Cell Transplantation for Severe Aplastic Anemia Using an Alemtuzumab-Containing Regimen Is Shaped by Persistence of Recipient CD8 T Cells.

Prevention of graft-versus-host disease (GVHD) is paramount for allogeneic hematopoietic stem cell transplantation (HSCT) to treat nonmalignant diseases. We previously reported that allogeneic HSCT for severe aplastic anemia (SAA) using the fludarabine, cyclophosphamide, and alemtuzumab (Campath-1H) (FCC) regimen is associated with a very low risk of GVHD and excellent clinical outcomes. We now report a single-center study of 45 patients with longer follow-up and investigation of lymphocyte recovery. Overall survival (OS) was 93%, and event-free survival (EFS) was 90.7%. Acute and chronic GVHD each occurred in 6 patients (13.3%), and only 1 case was severe. Mixed T cell chimerism was frequent and persisted after cessation of immunosuppression. T cells were extensively depleted, representing only 11.3% of lymphocytes at day 30 and rising to 43.8% by 1 year, but still significantly below normal levels (67.2%; P = .018), and deficiency persisted after immunosuppressive therapy (IST) withdrawal. Depletion of CD4 T cells was particularly profound, causing inversion of the normal CD4:CD8 T cell ratio. T cell subset composition was also abnormal, with memory and effector T cells predominating for at least 6 months after FCC HSCT. Analysis of T cell subset chimerism showed that CD4 T cells were predominantly donor-derived at 1 year, whereas recipient-derived CD8 T cells shaped mixed chimerism with a notable contribution of recipient effector CD8 T cells. The prolonged mixed T cell chimerism after IST withdrawal and low incidence of GVHD indicates the establishment of mutual tolerance, but the low incidence of viral disease suggests maintenance of antiviral immunity. Our study shows that despite the abnormal T cell profile after allogeneic HSCT for SAA using the FCC regimen, this regimen is conducive to an excellent clinical outcome.

Long-term labeling and imaging of synaptically connected neuronal networks in vivo using double-deletion-mutant rabies viruses.

Rabies-virus-based monosynaptic tracing is a widely used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Viral spread requires expression of both deleted viral genes in trans in postsynaptic source cells. Suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full 12-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis.

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry.

Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.

Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome.

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1-0.8) and 2.1 (P<0.04, 95% CI, 1.1-4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease evolution with emerging oncogenic mutations adversely affecting patients' outcome, implicating spliceosome mutations as founder mutations in myelodysplastic syndromes.

Third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency.

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here, we introduce third-generation (ΔL) rabies viral vectors, which appear to be as nontoxic as second-generation ones but have the major advantage of growing to much higher titers, resulting in significantly increased numbers of retrogradely labeled neurons in vivo.

Prevalence and clinical outcomes of the 46/1 haplotype, Janus kinase 2 mutations, and ten-eleven translocation 2 mutations in Budd-Chiari syndrome and their impact on thrombotic complications post liver transplantation.

Latent myeloproliferative disorders (MPDs) can be identified by Janus kinase 2 (JAK2) mutations in patients with idiopathic Budd-Chiari syndrome (BCS). The incidence and clinical outcomes of JAK2 mutations, novel ten-eleven translocation 2 (TET2) mutations, and the 46/1 haplotype in BCS are unknown for liver transplantation (LT). We undertook molecular studies of 66 patients presenting with BCS and correlated the results with the clinical outcomes. An overt MPD was present in 20% of the cases, and a latent MPD confirmed by the presence of a JAK2 mutation was detected in 45%. Testing for a TET2 mutation identified MPDs at the molecular level in another 7% of the subset of patients with BCS who were evaluated. The 46/1 haplotype frequency was significantly greater in BCS patients versus the general population (P < 0.001). The presence of JAK2 and TET2 mutations had no impact on 1-year survival. Thirty-six patients underwent LT, and 12 developed liver-related thrombotic complications (33%). Ten of these 12 patients required retransplantation. Retransplantation was more likely in those patients who developed liver-related thrombotic complications (P < 0.001). A JAK2 mutation was highly associated with the development of thrombotic complications after LT (P = 0.005). In conclusion, the presence of JAK2V617F predicts hepatic and extrahepatic thrombotic complications after LT. Testing for TET2 mutations can identify another 7% of idiopathic BCS patients with molecular MPDs.

Next-generation sequencing of the TET2 gene in 355 MDS and CMML patients reveals low-abundance mutant clones with early origins, but indicates no definite prognostic value.

Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using "next-generation" sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chronic myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. "Deeper" sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34(+) and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Anterior thalamic dysfunction underlies cognitive deficits in a subset of neuropsychiatric disease models.

Neuropsychiatric disorders are often accompanied by cognitive impairments/intellectual disability (ID). It is not clear whether there are converging mechanisms underlying these debilitating impairments. We found that many autism and schizophrenia risk genes are expressed in the anterodorsal subdivision (AD) of anterior thalamic nuclei, which has reciprocal connectivity with learning and memory structures. CRISPR-Cas9 knockdown of multiple risk genes selectively in AD thalamus led to memory deficits. While the AD is necessary for contextual memory encoding, the neighboring anteroventral subdivision (AV) regulates memory specificity. These distinct functions of AD and AV are mediated through their projections to retrosplenial cortex, using differential mechanisms. Furthermore, knockdown of autism and schizophrenia risk genes PTCHD1, YWHAG, or HERC1 from AD led to neuronal hyperexcitability, and normalization of hyperexcitability rescued memory deficits in these models. This study identifies converging cellular to circuit mechanisms underlying cognitive deficits in a subset of neuropsychiatric disease models.

Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations.

Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent "helper" adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.

Impact of somatic mutations in myelodysplastic patients with isolated partial or total loss of chromosome 7.

Monosomy 7 [-7] and/or partial loss of chromosome 7 [del(7q)] are associated with poor and intermediate prognosis, respectively, in myelodysplastic syndromes (MDS), but somatic mutations may also play a key complementary role. We analyzed the impact on the outcomes of deep targeted mutational screening in 280 MDS patients with -7/del(7q) as isolated cytogenetic abnormality (86 with del(7q) and 194 with -7). Patients with del(7q) or -7 had similar demographic and disease-related characteristics. Somatic mutations were detected in 79% (93/117) of patients (82% in -7 and 73% in del(7q) group). Median number of mutations per patient was 2 (range 0-8). There was no difference in mutation frequency between the two groups. Patients harbouring ≥2 mutations had a worse outcome than patients with <2 or no mutations (leukaemic transformation at 24 months, 38% and 20%, respectively, p = 0.044). Untreated patients with del(7q) had better overall survival (OS) compared with -7 (median OS, 34 vs 17 months, p = 0.034). In multivariable analysis, blast count, TP53 mutations and number of mutations were independent predictors of OS, whereas the cytogenetic subgroups did not retain prognostic relevance. This study highlights the importance of mutational analysis in terms of prognosis in MDS patients with isolated -7 or del(7q).

Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes.

UNLABELLED: Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors. DESIGN AND METHODS: Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents. RESULTS: PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.

p27Kip1 and p130 cooperate to regulate hematopoietic cell proliferation in vivo.

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.

Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.

Effect of low-level BCR-ABL1 kinase domain mutations identified by next-generation sequencing in patients with chronic myeloid leukaemia: a population-based study.

BACKGROUND: Kinase domain mutations in BCR-ABL1 are associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukaemia. Next-generation sequencing (NGS) allows detection of low-level kinase domain mutations, but its relevance in clinical practice remains debated. We aimed to examine the clinical effects of low-level kinase domain mutations identified using NGS in patients with chronic myeloid leukaemia. METHODS: In this population-based study, we included consecutive patients newly diagnosed with chronic myeloid leukaemia treated with first-line tyrosine kinase inhibitors, and patients identified at the time of resistance to first-line treatment with imatinib at six institutions (teaching hospitals and district hospitals) in southeast England. We screened patients for BCR-ABL1 kinase domain mutations using NGS, irrespective of patient response to tyrosine kinase inhibitor therapy. When we detected a mutation with NGS, we retrospectively analysed all previous samples to establish the date of first occurrence and subsequent kinetics of the mutant subclone (or subclones). The primary endpoints of this study were progression-free and event-free survival at 5 years. FINDINGS: Between Feb 1, 2007, and Dec 31, 2014, we screened 121 patients with chronic myeloid leukaemia for BCR-ABL1 kinase domain mutation. 99 consecutive patients were newly diagnosed, with available sequential RNA stored. The remaining 22 patients were diagnosed between June 1, 1999, and June 30, 2006, and were screened at the time of resistance to first-line treatment with imatinib. Imatinib was the first-line treatment for 111 patients, nilotinib for seven patients, and dasatinib for three patients. We detected a kinase domain mutation in 25 (21%) of 121 patients. Low-level kinase domain mutations were first identified in 17 (68%) of 25 patients with mutation. For patients with a complete cytogenetic response, 13 (14%) of 93 patients screened had a mutation. Five (71%) of the seven patients with a clinically relevant mutation lost complete cytogenetic response compared with 15 (17%) of 86 patients without a clinically relevant mutation (80 patients without mutation and six patients with a tyrosine kinase inhibitor-sensitive mutation, p=0·0031). Patients harbouring a mutant clone had poorer 5-year progression-free survival (65·3% [95% CI 40·5-81·8] vs 86·9% [75·8-93·2]; p=0·0161) and poorer 5-year event-free survival (22·2% [CI 5·6-45·9] vs 62·0% [50·4-71·6]; p<0·0001) than did patients without a mutation. We identified a kinase domain mutation in four (10%) of 41 patients with samples available at 3 months after starting first-line tyrosine kinase inhibitor treatment; all four subsequently progressed to accelerated phase disease compared with only three (8%) of 37 without a mutation (p<0·0001). INTERPRETATION: NGS reliably and consistently detected early appearance of kinase domain mutations that would not otherwise be detected by Sanger sequencing. For the first time, to our knowledge, we report the presence of kinase domain mutations after only 3 months of therapy, which could have substantial clinical implications. NGS will allow early clinical intervention and our findings will contribute to the establishment of new recommendations on the frequency of kinase domain mutation analysis to improve patient clinical care. FUNDING: None.

AKT1 (E17K) mutation in pancreatic cancer.

The prevalence of a novel somatic mutation (E17K) in the pleckstrin homology domain of AKT1 was investigated in pancreatic cancer using a quantitative pyrosequencing assay. This mutation was un-detectable in pancreatic cancer tissue samples (n=65) and pancreatic cell line (n=10) DNA suggesting that pancreatic cancer progression is mainly dependent on the K-Ras mutation.