Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells

Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin- Hoechst(low)/Rhodamine(low) (Lin- Ho(low)/Rho(low)) and 10% to 15% of Lin-/Sca+ cells in S phase. These cells enter proliferative cycle and progress through G1 and into S phase in the presence of cytokines and 5% heat-inactivated fetal calf serum (HI-FCS). Cytokine-stimulated Lin- Ho(low)/Rho(low) cells took 36 to 40 hours to complete first division and only 12 hours to complete each of 5 subsequent divisions. These cells require 16 to 18 hours to transit through G0/G1 period and 28 to 30 hours to enter into mid-S phase during the first cycle. Up to 56% of Lin- Rho(low)/Ho(low) cells are high-proliferative potential (7 factor-responsive) colony-forming cells (HPP-CFC). At isolation, HPP-CFC are quiescent, but after 28 to 30 hours of culture, greater than 60% are in S phase. Isoleucine-deprivation of Lin- Ho(low)/Rho(low) cells in S phase of first cycle reversibly blocked them from entering into second cycle. After the release from isoleucine-block, these cells exhibited a G1 period of less than 2 hours and entered into mid-S phase by 12 hours. Thus, the duration of G1 phase of the cells in second cycle is 4 to 5 times shorter than that observed in their first cycle. Similar cell cycle kinetics are observed with Lin-/Sca+ population of bone marrow cells. Stem cell factor (SCF) alone, in the presence of HI-FCS, is as effective as a cocktail of 2 to 7 cytokines in inducing quiescent Lin-/Sca+ cells to enter into proliferative cycle. Aphidicolin treatment reversibly blocked cytokine-stimulated Lin-/Sca+ cells at G1/S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G1. For these cells also, SCF alone is sufficient for their progression through S phase. These studies indicate a very short G1 phase for stem cells induced to proliferate and offer experimental approaches to synchronize murine hematopoietic stem cells

Mild motor impairment as prodromal state in amyotrophic lateral sclerosis:A new diagnostic entity

Amyotrophic lateral sclerosis, when viewed as a biological entity rather than a clinical syndrome, probably evolves along a continuum, with the initial clinically silent phase eventually evolving into clinically manifest amyotrophic lateral sclerosis. Since motor neuron degeneration is incremental and cumulative over time, it stands to reason that the clinical syndrome of amyotrophic lateral sclerosis is probably preceded by a prodromal state characterized by minor motor abnormalities that are initially insufficient to permit a diagnosis of amyotrophic lateral sclerosis. This prodromal period, however, is usually missed, given the invariably long delays between symptom onset and diagnostic evaluation. The Pre-Symptomatic Familial ALS Study, a cohort study of pre-symptomatic gene mutation carriers, offers a unique opportunity to observe what is typically unseen. Here we describe the clinical characterization of 20 pre-symptomatic mutation carriers (in SOD1, FUS and C9orf72) whose phenoconversion to clinically manifest disease has been prospectively studied. In so doing, we observed a prodromal phase of mild motor impairment in 11 of 20 phenoconverters. Among the n = 12 SOD1 A4V mutation carriers, phenoconversion was characterized by abrupt onset of weakness, with a short (1–3.5 months) prodromal period observable in a small minority (n = 3); the observable prodrome invariably involved the lower motor neuron axis. By contrast, in all n = 3 SOD1 I113T mutation carriers, diffuse lower motor neuron and upper motor neuron signs evolved insidiously during a prodromal period that extended over a period of many years; prodromal manifestations eventually coalesced into a clinical syndrome that is recognizable as amyotrophic lateral sclerosis. Similarly, in all n = 3 C9orf72 hexanucleotide repeat expansion mutation carriers, focal or multifocal manifestations of disease evolved gradually over a prodromal period of 1–2 years. Clinically manifest ALS also emerged following a prodromal period of mild motor impairment, lasting >4 years and ∼9 months, respectively, in n = 2 with other gene mutations (SOD1 L106V and FUS c.521del6). On the basis of this empirical evidence, we conclude that mild motor impairment is an observable state that precedes clinically manifest disease in three of the most common genetic forms of amyotrophic lateral sclerosis (SOD1, FUS, C9orf72), and perhaps in all genetic amyotrophic lateral sclerosis; we also propose that this might be true of non-genetic amyotrophic lateral sclerosis. As a diagnostic label, mild motor impairment provides the language to describe the indeterminate (and sometimes intermediate) transition between the unaffected state and clinically manifest amyotrophic lateral sclerosis. Recognizing mild motor impairment as a distinct clinical entity should generate fresh urgency for developing biomarkers reflecting the earliest events in the degenerative cascade, with potential to reduce the diagnostic delay and to permit earlier therapeutic intervention

An endogenous peptide marker differentiates SOD1 stability and facilitates pharmacodynamic monitoring in SOD1 amyotrophic lateral sclerosis

The discovery of novel biomarkers has emerged as a critical need for therapeutic development in amyotrophic lateral sclerosis (ALS). For some subsets of ALS, such as the genetic superoxide dismutase 1 (SOD1) form, exciting new treatment strategies, such as antisense oligonucleotide-mediated (ASO-mediated) SOD1 silencing, are being tested in clinical trials, so the identification of pharmacodynamic biomarkers for therapeutic monitoring is essential. We identify increased levels of a 7-amino acid endogenous peptide of SOD1 in cerebrospinal fluid (CSF) of human SOD1 mutation carriers but not in other neurological cases or nondiseased controls. Levels of peptide elevation vary based on the specific SOD1 mutation (ranging from 1.1-fold greater than control in D90A to nearly 30-fold greater in V148G) and correlate with previously published measurements of SOD1 stability. Using a mass spectrometry-based method (liquid chromatography-mass spectrometry), we quantified peptides in both extracellular samples (CSF) and intracellular samples (spinal cord from rat) to demonstrate that the peptide distinguishes mutation-specific differences in intracellular SOD1 degradation. Furthermore, 80% and 63% reductions of the peptide were measured in SOD1G93A and SOD1H46R rat CSF samples, respectively, following treatment with ASO, with an improved correlation to mRNA levels in spinal cords compared with the ELISA measuring intact SOD1 protein. These data demonstrate the potential of this peptide as a pharmacodynamic biomarker

Randomized, double-blind, placebo-controlled trial of arimoclomol in rapidly progressive SOD1 ALS

Objective: To examine the safety and tolerability as well as the preliminary efficacy of arimoclomol, a heat shock protein co-inducer that promotes nascent protein folding, in patients with rapidly progressive SOD1 amyotrophic lateral sclerosis (ALS). Methods: This was a double-blind, placebo-controlled trial in which patients with rapidly progressive SOD1-mutant ALS were randomized 1:1 to receive arimoclomol 200 mg tid or matching placebo for up to 12 months. Study procedures were performed using a mix of in-person and remote assessments. Primary outcome was safety and tolerability. Secondary outcome was efficacy, with survival as the principal measure. Additional efficacy measures were the rates of decline of the Revised ALS Functional Rating Scale (ALSFRS-R) and percent predicted forced expiratory volume in 6 seconds (FEV6), and the Combined Assessment of Function and Survival (CAFS). Results: Thirty-eight participants were randomized. Thirty-six (19 placebo, 17 arimoclomol) were included in the prespecified intent-to-treat analysis. Apart from respiratory function, groups were generally well-balanced at baseline. Adverse events occurred infrequently, and were usually mild and deemed unlikely or not related to study drug. Adjusting for riluzole and baseline ALSFRS-R, survival favored arimoclomol with a hazard ratio of 0.77 (95% confidence interval [CI] 0.32–1.80). ALSFRS-R and FEV6 declined more slowly in the arimoclomol group, with treatment differences of 0.5 point/month (95% CI −0.63 to 1.63) and 1.24 percent predicted/month (95% CI −2.77 to 5.25), respectively, and the CAFS similarly favored arimoclomol. Conclusions: This study provides Class II evidence that arimoclomol is safe and well-tolerated at a dosage of 200 mg tid for up to 12 months. Although not powered for therapeutic effect, the consistency of results across the range of prespecified efficacy outcome measures suggests a possible therapeutic benefit of arimoclomol. Clinicaltrials.gov identifier NCT00706147. Classification of evidence This study provides Class II evidence that arimoclomol is safe and well-tolerated at a dosage of 200 mg tid for up to 12 months. The study lacked the precision to conclude, or to exclude, an important therapeutic benefit of arimoclomol

Temporal course of cognitive and behavioural changes in motor neuron diseases

Background Cognitive and behavioural dysfunction may occur in people with motor neuron disease (MND), with some studies suggesting an association with the C9ORF72 repeat expansion. Their onset and progression, however, is poorly understood. We explored how cognition and behaviour change over time, and whether demographic, clinical and genetic factors impact these changes. Methods Participants with MND were recruited through the Phenotype-Genotype-Biomarker study. Every 3–6 months, the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) was used to assess amyotrophic lateral sclerosis (ALS) specific (executive functioning, verbal fluency, language) and ALS non-specific (memory, visuospatial) functions. Informants reported on behaviour symptoms via semi-structured interview. Results Participants with neuropsychological data at ≥3 visits were included (n=237, mean age=59, 60% male), of which 18 (8%) were C9ORF72 positive. Baseline cognitive impairment was apparent in 18 (8%), typically in ALS specific domains, and associated with lower education, but not C9ORF72 status. Cognition, on average, remained stable over time, with two exceptions: (1) C9ORF72 carriers declined in all ECAS domains, (2) 8%–9% of participants with baseline cognitive impairment further declined, primarily in the ALS non-specific domain, which was associated with less education. Behavioural symptoms were uncommon. Conclusions In this study, cognitive dysfunction was less common than previously reported and remained stable over time for most. However, cognition declines longitudinally in a small subset, which is not entirely related to C9ORF72 status. Our findings raise questions about the timing of cognitive impairment in MND, and whether it arises during early clinically manifest disease or even prior to motor manifestations

Urinary p75(ECD): A prognostic, disease progression, and pharmacodynamic biomarker in ALS.

OBJECTIVE: To evaluate urinary neurotrophin receptor p75 extracellular domain (p75(ECD)) levels as disease progression and prognostic biomarkers in amyotrophic lateral sclerosis (ALS). METHODS: The population in this study comprised 45 healthy controls and 54 people with ALS, 31 of whom were sampled longitudinally. Urinary p75(ECD) was measured using an enzyme-linked immunoassay and validation included intra-assay and inter-assay coefficients of variation, effect of circadian rhythm, and stability over time at room temperature, 4°C, and repeated freeze-thaw cycles. Longitudinal changes in urinary p75(ECD) were examined by mixed model analysis, and the prognostic value of baseline p75(ECD) was explored by survival analysis. RESULTS: Confirming our previous findings, p75(ECD) was higher in patients with ALS (5.6 ± 2.2 ng/mg creatinine) compared to controls (3.6 ± 1.4 ng/mg creatinine, p < 0.0001). Assay reproducibility was high, with p75(ECD) showing stability across repeated freeze-thaw cycles, at room temperature and 4°C for 2 days, and no diurnal variation. Urinary p75(ECD) correlated with the revised ALS Functional Rating Scale at first evaluation (r = -0.44, p = 0.008) and across all study visits (r = -0.36, p < 0.0001). p75(ECD) also increased as disease progressed at an average rate of 0.19 ng/mg creatinine per month (p < 0.0001). In multivariate prognostic analysis, bulbar onset (hazard ratio [HR] 3.0, p = 0.0035), rate of disease progression from onset to baseline (HR 4.4, p < 0.0001), and baseline p75(ECD) (HR 1.3, p = 0.0004) were predictors of survival. CONCLUSIONS: The assay for urinary p75(ECD) is analytically robust and shows promise as an ALS biomarker with prognostic, disease progression, and potential pharmacodynamic application. Baseline urinary p75(ECD) provides prognostic information and is currently the only biological fluid-based biomarker of disease progression

The Fluctuating Phenotype of the Lymphohematopoietic Stem Cell with Cell Cycle Transit

The most primitive engrafting hematopoietic stem cell has been assumed to have a fixed phenotype, with changes in engraftment and renewal potential occurring in a stepwise irreversible fashion linked with differentiation. Recent work shows that in vitro cytokine stimulation of murine marrow cells induces cell cycle transit of primitive stem cells, taking 40 h for progression from G0 to mitosis and 12 h for subsequent doublings. At 48 h of culture, progenitors are expanded, but stem cell engraftment is markedly diminished. We have investigated whether this effect on engraftment was an irreversible step or a reversible plastic feature correlated with cell cycle progression. Long-term engraftment (2 and 6 mo) of male BALB/c marrow cells exposed in vitro to interleukin (IL)-3, IL-6, IL-11, and steel factor was assessed at 2–4-h intervals of culture over 24–48 h using irradiated female hosts; the engraftment phenotype showed marked fluctuations over 2–4-h intervals, with engraftment nadirs occurring in late S and early G2. These data show that early stem cell regulation is cell cycle based, and have critical implications for strategies for stem cell expansion and engraftment or gene therapy, since position in cell cycle will determine whether effective engraftment occurs in either setting

Abnormal Expression Of Homeobox Genes And Transthyretin In C9Orf72 Expansion Carriers

Objective: We performed a genome-wide brain expression study to reveal the underpinnings of diseases linked to a repeat expansion in chromosome 9 open reading frame 72 (C9ORF72). Methods: The genome-wide expression profile was investigated in brain tissue obtained from C9ORF72 expansion carriers (n = 32), patients without this expansion (n = 30), and controls (n = 20). Using quantitative real-time PCR, findings were confirmed in our entire pathologic cohort of expansion carriers (n = 56) as well as nonexpansion carriers (n = 31) and controls (n = 20). Results: Our findings were most profound in the cerebellum, where we identified 40 differentially expressed genes, when comparing expansion carriers to patients without this expansion, including 22 genes that have a homeobox (e.g., HOX genes) and/or are located within the HOX gene cluster (top hit: homeobox A5 [HOXA5]). In addition to the upregulation of multiple homeobox genes that play a vital role in neuronal development, we noticed an upregulation of transthyretin (TTR), an extracellular protein that is thought to be involved in neuroprotection. Pathway analysis aligned with these findings and revealed enrichment for gene ontology processes involved in (anatomic) development (e.g., organ morphogenesis). Additional analyses uncovered that HOXA5 and TTR levels are associated with C9ORF72 variant 2 levels as well as with intron-containing transcript levels, and thus, disease-related changes in those transcripts may have triggered the upregulation of HOXA5 and TTR. Conclusions: In conclusion, our identification of genes involved in developmental processes and neuroprotection sheds light on potential compensatory mechanisms influencing the occurrence, presentation, and/or progression of C9ORF72-related diseases

Phosphorylated neurofilament heavy chain : a biomarker of survival for C9ORF72-associated amyotrophic lateral sclerosis

As potential treatments for C9ORF72-associated amyotrophic lateral sclerosis (c9ALS) approach clinical trials, the identification of prognostic biomarkers for c9ALS becomes a priority. We show that levels of phosphorylated neurofilament heavy chain (pNFH) in cerebrospinal fluid (CSF) predict disease status and survival in c9ALS patients, and are largely stable over time. Moreover, c9ALS patients exhibit higher pNFH levels, more rapid disease progression, and shorter survival after disease onset than ALS patients without C9ORF72 expansions. These data support the use of CSF pNFH as a prognostic biomarker for clinical trials, which will increase the likelihood of successfully developing a treatment for c9ALS