Thomas J. Langan

Cell Cycle‐Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/ vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose‐dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein‐poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low‐density lipoprotein (LDL) to raise the cholesterol concentration 80‐fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more. This temporal aspect of the requirement for mevalonate was unaffected by the presence of a 90‐fold excess of cholesterol, provided as LDL. Thus, these data establish a requirement for mevalonate or for a presumably nonsterol derivative of mevalonate for the occurrence of DNA synthesis in synchronized glial primary cultures, and demonstrate that this requirement is expressed at a specific time in the cell cycle.

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Abstract: Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3‐hydroxy‐3‐methylglutaryl‐CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24‐h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.

Synchronization of Mammalian Cell Cultures by Serum Deprivation

Mammalian cells are amenable to study the regulation of cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is applicable to other mammalian cells. Its application allows astrocytes to reenter the cell cycle from a state of quiescence (G(0)), and then, under carefully defined experimental conditions, to move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include physical separation by centrifugal elutriation and mitotic shake off or chemically induced cell cycle arrest. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum downshift and arrested in the G(0) phase of the cell cycle. This procedure has been extended to the examination of cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

Clinical outcomes of children with abnormal newborn screening results for Krabbe disease in New York State

Background:Early infantile Krabbe disease is rapidly fatal, but hematopoietic stem cell transplantation (HSCT) may improve outcomes if performed soon after birth. New York State began screening all newborns for Krabbe disease in 2006.Methods:Infants with abnormal newborn screen results for Krabbe disease were referred to specialty-care centers. Newborns found to be at high risk for Krabbe disease underwent a neurodiagnostic battery to determine the need for emergent HSCT.Results:Almost 2 million infants were screened. Five infants were diagnosed with early infantile Krabbe disease. Three died, two from HSCT-related complications and one from untreated disease. Two children who received HSCT have moderate to severe developmental delays. Forty-six currently asymptomatic children are considered to be at moderate or high risk for development of later-onset Krabbe disease.Conclusions:These results show significant HSCT-associated morbidity and mortality in early infantile Krabbe disease and raise questions about its efficacy when performed in newborns diagnosed through newborn screening. The unanticipated identification of “at risk” children introduces unique ethical and medicolegal issues. New York’s experience raises questions about the risks, benefits, and practicality of screening newborns for Krabbe disease. It is imperative that objective assessments be made on an ongoing basis as additional states begin screening for this disorder.Genet Med 18 12, 1235–1243.

Astrocytes derived from long-term primary cultures recapitulate features of astrogliosis as they re-enter the cell division cycle

We investigated whether the initiation of cell cycling by astrocytes after prolonged quiescence in long-term primary cultures is associated with immunocytochemical changes that characterize reactive astrogliosis. Primary cultures of newborn rat brain were maintained for greater than 2 months in a stable quiescent state. Partially synchronous transition through a single cell cycle was achieved by trypsinization and replating, and then after 2-3 days, by 48 h of serum depletion and serum shift-up to 10% (time 0). At time 0, the percentages of cells decorated by monoclonal antibodies specific for bromodeoxyuridine (BrDU) after a 2.5 h pulse, and for glial fibrillary acidic protein (GFAP) and vimentin (VIM) were respectively 8 +/- 2, 15 +/- 4 and 10 +/- 3. By 24 h (S phase), 64 +/- 7% of nuclei were (BrDU+), and percentages of (GFAP+) and (VIM+) cells were 19 +/- 4 and 87 +/- 12, respectively. Dual label immunofluorescence showed that greater than 75% of (GFAP+) cells were indeed (VIM+/GFAP+) at 24 h, and that the percentages of (VIM+), (BrDU+) and (VIM+/BrDU+) cells were equivalent for the duration of the first cell cycle (36-48 h). By 72-96 h, (VIM+) cells decreased to less than 10%, and (BrDU+) cells numbered 32 +/- 8%, while (GFAP+) cells increased to around 90%. Ran-2 immunofluorescence at 96 h identified virtually all of the cells as type 1 astrocytes. Thus, astrocytes after months of quiescence give rise to cells that recapitulate a VIM/GFAP transition in a manner resembling astrogliosis, and do so in relation to progression through a single cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Quiescent astroglia in long-term primary cultures re-enter the cell cycle and require a non-sterol isoprenoid in late G1

Astroglia proliferate during brain growth, and can divide again later, particularly during astrogliosis. We investigated whether astroglia in primary cultures of newborn rat brain similarly achieve a state of prolonged quiescence which enables re-entry into the cell division cycle. In cultures after 2 months, cell number plateaued and there were sharp decreases in [3H]thymidine incorporation (70 +/- 5 vs 4 +/- 0.5 cpm/micrograms protein/h at 30 and 60 days, respectively) and in percentages of cell nuclei incorporating bromodeoxyuridine (BrDU) (from 46 +/- 6% to less than 1%). Replating at 10(4) cells/cm2 yielded secondary cultures which synthesized DNA actively. Forty-eight hours of serum deprivation at 2-3 days from subculturing, followed by addition of 10% serum (time 0), resulted in a return to quiescence which persisted until 12 h (G0 + G1). By 20 h (S phase), there were abrupt increases in DNA synthesis (5-fold) and in BrDU-labeled nuclei (from 19 +/- 2 to 76 +/- 8%) and the percentage of glial fibrillary acidic protein (GFAP)-positive cells declined to 14 +/- 2%. Three days later, GFAP-positive cells numbered around 80%. Cell cycling after prolonged quiescence, in a manner similar to that in early astroglial cultures, required a non-sterol derivative of mevalonate in late G1. These data confirm that astroglia in primary cultures, like their counterparts in vivo, have a flexible capacity to enter and depart from quiescence, and most importantly, provide a system for examining regulation of this process.

In vitro synchronization of mammalian astrocytic cultures by serum deprivation.

The study of the regulation of cell division cycle in vitro requires cell cultures growing in the same phase of the cycle. The procedure by which cells are arrested in specific phases of the cell cycle is termed synchronization. Synchronization is particularly important in the study of astrocyte biology, as its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G(0)), and, under carefully defined experimental conditions, move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, including centrifugal elutriation, mitotic shake-off, and chemically induced cell cycle arrest. Yet there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocytic cultures from newborn rat brains by serum deprivation. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum down-shift and arrested in the G(0) phase of the cell cycle. This procedure has recently been extended toward the study of cell cycle control in astroglioma cells and astrocytes from injured adult brains. Since it was also employed in recent precursor cloning studies in developmental biology, this procedure will certainly find increasing use in future research.

Neurologic complications of 2009 influenza-a H1N1 infection in children.

Little is known about the neurologic complications of the 2009 Influenza-A H1N1 epidemic in children. We present a retrospective analysis of children evaluated at a tertiary children’s hospital who tested positive for H1N1 with neurologic complications. A total of 164 children tested positive for H1N1. Thirty-one of these patients (19%) were evaluated and discharged from the emergency department. Thirty-nine (24%) were treated in the intensive care unit, the remaining 94 (57%) were treated in medical in-patient units. Six subjects died (3.7%). Neurologic complications identified included headache, encephalitis, polyneuropathy, seizures, and malignant hyperthermia. The rate of neurologic complications in this cohort of patients who tested positive for H1N1 was 19%. The incidence of serious neurologic complications was 3%, with another 3% of patients who experienced rapid clinical deterioration and subsequently died. Our observation of neurologic complications associated with 2009 influenza-A H1N1 epidemic suggests the need for clinical vigilance during future influenza epidemics.

Cell cycle kinetics and commitment in newborn, adult, and tumoral astrocytes

In terms of cell cycle phases, mammalian astrocytes maintain the capacity to leave G0/G1 and enter S phase in response to brain injury or due to neoplastic transformation. This report compares proliferative behavior in vitro, particularly departure from G0, in three types of rat astroglial cells-newborn astrocytes, astrocytes from gelatin implants into the traumatized striata of adults, and astrocytoma cells (C6 glioma). Newborn and adult astrocytes demonstrated nearly identical proliferation kinetics as determined by peaks in cell number and rates of DNA synthesis. C6 glioma (C6G) proliferated more rapidly. Exit from G0 was examined by shift-down of serum from 10 to 0.1% for 48 h, followed by return to 10% at time 0. Synchronization of newborn and adult astrocytes in this way resulted in a 12 h lag phase (G0/G1) followed by a 6-10-fold surge in DNA synthesis and a corresponding increase in S-phase nuclei from < 15% to > 70%. Timing of S-phase commitment was established in late G1 by resistance to the inhibitors cycloheximide and mevinolin. Decay of commitment was assessed by addition of hydroxyurea (HU) at 10 h to cause accumulation at the G1/S boundary. Removal of HU after an additional 14, 16, and 20 h resulted respectively in these percentages of maximal S-phase DNA synthesis in newborn and adult astrocytes: 75 +/- 9, 60 +/- 8, 23 +/- 3, and 87 +/- 20, 62 +/- 7, 34 +/- 5. In contrast, synchronization of C6G resulted in a 6 h lag before a surge in DNA synthesis and an increase in S-phase nuclei from < 20% to 100%. Cell cycle commitment occurred earlier with C6G, and decay of commitment was not observed, even after 20 h of HU treatment. Thus, these in vitro techniques for cell cycle analysis are applicable to astrocytes obtained from developing and adult brain, and to at least some astroglioma cells. Furthermore, this comparative study showed that important cell cycle parameters differ markedly in the non-tumoral astrocytes and glioma cells. These differences could lead to strategies for selective targeting of the proliferation of neoplastic astroglia.

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

Abstract: The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum‐depleted (0.1%, vol/vol) for 48 h on days 4–6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70–80% of the cells were glial fibrillary acidic protein (GFAP)‐negative by indirect immunofluorescence; 79 ± 7% were GFAP‐positive after an additional 3 days. Serum shift‐up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 ± 6% to 75 ± 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 ± 6 versus 380 ± 32 cpm/μg of protein/h of [3H]thymidine uptake). Addition of mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 μM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine‐labeled nuclei increased from 0% to 75 ± 9%, and DNA synthesis increased 10‐fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.