Pushing myelination - developmental regulation of myosin expression drives oligodendrocyte morphological differentiation.

Oligodendrocytes are the central nervous system myelin-forming cells providing axonal electrical insulation and higher-order neuronal circuitry. The mechanical forces driving the differentiation of oligodendrocyte precursor cells into myelinating oligodendrocytes are largely unknown, but likely require the spatiotemporal regulation of the architecture and dynamics of the actin and actomyosin cytoskeletons. In this study, we analyzed the expression pattern of myosin motors during oligodendrocyte development. We report that oligodendrocyte differentiation is regulated by the synchronized expression and non-uniform distribution of several members of the myosin network, particularly non-muscle myosins 2B and 2C, which potentially operate as nanomechanical modulators of cell tension and myelin membrane expansion at different cell stages.This article has an associated First Person interview with the first author of the paper.

Combinatorial actions of Tgfß and Activin ligands promote oligodendrocyte development and CNS myelination.

In the embryonic CNS, development of myelin-forming oligodendrocytes is limited by bone morphogenetic proteins, which constitute one arm of the transforming growth factor-ß (Tgfß) family and signal canonically via Smads 1/5/8. Tgfß ligands and Activins comprise the other arm and signal via Smads 2/3, but their roles in oligodendrocyte development are incompletely characterized. Here, we report that Tgfß ligands and activin B (ActB) act in concert in the mammalian spinal cord to promote oligodendrocyte generation and myelination. In mouse neural tube, newly specified oligodendrocyte progenitors (OLPs) are first exposed to Tgfß ligands in isolation, then later in combination with ActB during maturation. In primary OLP cultures, Tgfß1 and ActB differentially activate canonical Smad3 and non-canonical MAP kinase signaling. Both ligands enhance viability, and Tgfß1 promotes proliferation while ActB supports maturation. Importantly, co-treatment strongly activates both signaling pathways, producing an additive effect on viability and enhancing both proliferation and differentiation such that mature oligodendrocyte numbers are substantially increased. Co-treatment promotes myelination in OLP-neuron co-cultures, and maturing oligodendrocytes in spinal cord white matter display strong Smad3 and MAP kinase activation. In spinal cords of ActB-deficient Inhbb(-/-) embryos, apoptosis in the oligodendrocyte lineage is increased and OLP numbers transiently reduced, but numbers, maturation and myelination recover during the first postnatal week. Smad3(-/-) mice display a more severe phenotype, including diminished viability and proliferation, persistently reduced mature and immature cell numbers, and delayed myelination. Collectively, these findings suggest that, in mammalian spinal cord, Tgfß ligands and ActB together support oligodendrocyte development and myelin formation.

MLCK regulates Schwann cell cytoskeletal organization, differentiation and myelination.

Signaling through cyclic AMP (cAMP) has been implicated in the regulation of Schwann cell (SC) proliferation and differentiation. In quiescent SCs, elevation of cAMP promotes the expression of proteins associated with myelination such as Krox-20 and P0, and downregulation of markers associated with the non-myelinating SC phenotype. We have previously shown that the motor protein myosin II is required for the establishment of normal SC-axon interactions, differentiation and myelination, however, the mechanisms behind these effects are unknown. Here we report that the levels and activity of myosin light chain kinase (MLCK), an enzyme that regulates MLC phosphorylation in non-muscle cells, are dramatically downregulated in SCs after cAMP treatment, in a similar pattern to that of c-Jun, a known inhibitor of myelination. Knockdown of MLCK in SCs mimics the effect of cAMP elevation, inducing plasma membrane expansion and expression of Krox-20 and myelin proteins. Despite activation of myelin gene transcription these cells fail to make compact myelin when placed in contact with axons. Our data indicate that myosin II activity is differentially regulated at various stages during myelination and that in the absence of MLCK the processes of SC differentiation and compact myelin assembly are uncoupled.

Atomic Force Microscopy-Based Measurements of Retinal Microvessel Stiffness in Mice with Endothelial-Specific Deletion of CCN1.

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.

Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1.

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.

Acute and chronic demyelinated CNS lesions exhibit opposite elastic properties.

Increased deposition of extracellular matrix (ECM) is a known inhibitor of axonal regrowth and remyelination. Recent in vitro studies have demonstrated that oligodendrocyte differentiation is impacted by the physical properties of the ECM. However, characterization of the mechanical properties of the healthy and injured CNS myelin is challenging, and has largely relied on non-invasive, low-resolution methods. To address this, we have employed atomic force microscopy to perform micro-indentation measurements of demyelinated tissue at cellular scale. Analysis of mouse and human demyelinated brains indicate that acute demyelination results in decreased tissue stiffness that recovers with remyelination; while chronic demyelination is characterized by increased tissue stiffness, which correlates with augmented ECM deposition. Thus, changes in the mechanical properties of the acutely (softer) or chronically (stiffer) demyelinated brain might contribute to differences in their regenerative capacity. Our findings are relevant to the optimization of cell-based therapies aimed at promoting CNS regeneration and remyelination.

Utility of the heteroduplex assay (HDA) as a simple and cost-effective tool for the identification of HIV type 1 dual infections in resource-limited settings.

The predominance of unique recombinant forms (URFs) of HIV-1 in Cameroon suggests that dual infection, the concomitant or sequential infection with genetically distinct HIV-1 strains, occurs frequently in this region; yet, identifying dual infection among large HIV cohorts in local, resource-limited settings is uncommon, since this generally relies on labor-intensive and costly sequencing methods. Consequently, there is a need to develop an effective, cost-efficient method appropriate to the developing world to identify these infections. In the present study, the heteroduplex assay (HDA) was used to verify dual or single infection status, as shown by traditional sequence analysis, for 15 longitudinally sampled study subjects from Cameroon. Heteroduplex formation, indicative of a dual infection, was identified for all five study subjects shown by sequence analysis to be dually infected. Conversely, heteroduplex formation was not detectable for all 10 HDA reactions of the singly infected study subjects. These results suggest that the HDA is a simple yet powerful and inexpensive tool for the detection of both intersubtype and intrasubtype dual infections, and that the HDA harbors significant potential for reliable, high-throughput screening for dual infection. As these infections and the recombinants they generate facilitate leaps in HIV-1 evolution, and may present major challenges for treatment and vaccine design, this assay will be critical for monitoring the continuing pandemic in regions of the world where HIV-1 viral diversity is broad.

A heteroduplex assay for the rapid detection of dual Human Immunodeficiency Virus Type 1 infections.

The predominance of circulating and unique recombinant forms (URFs) of Human Immunodeficiency Virus Type 1 (HIV-1) in Cameroon suggests that dual infection occurs frequently in this region. Despite the potential impact of these infections on the evolution of HIV diversity, relatively few have been detected. The failure to detect dual infections may be attributable to the laborious and costly sequence analysis involved in their identification. As such, there is a need for a cost-effective, more rapid method to efficiently distinguish this subset of HIV-positive individuals, particularly in regions where HIV diversity is broad. In the present study, the heteroduplex assay (HDA) was developed to detect dual HIV-1 infection. This assay was validated on sequential specimens obtained from 20 HIV+ study subjects, whose single or dual infection status was determined by standard sequence analysis. By mixing gag fragments amplified from the sequential specimens from each study subject in HDA reactions, it was shown that single and dual infection status correlated with the absence and presence, respectively, of heteroduplex bands upon gel electrophoresis. Therefore, this novel assay is capable of identifying dual infections with a sensitivity and specificity equivalent to that of sequence analysis. Given the impact of dual infection on viral recombination and diversity, this simple technique will be beneficial to understanding HIV-1 evolution within an individual, as well as at a population level, in West-Central Africa and globally.

Preparation of Matrices of Variable Stiffness for the Study of Mechanotransduction in Schwann Cell Development.

Extracellular matrix (ECM) elasticity may direct cellular differentiation and can be modeled in vitro using synthetic ECM-like substrates with defined elastic properties. However, the effectiveness of such approaches depends on the selection of a range of elasticity and ECM ligands that accurately model the relevant tissue. Here, we present a cell culture system than can be used to study Schwann cell differentiation on substrates which model the changes in mechanical ECM properties that occur during sciatic nerve development.

Circulating recombinant form (CRF) 37_cpx: an old strain in Cameroon composed of diverse, genetically distant lineages of subtypes A and G.

HIV-1 in Cameroon is genetically diverse, but is predominated by the circulating recombinant form (CRF) 02_AG, which cocirculates among an array of other CRFs, unique recombinant forms (URFs), and all group M subtypes. In particular, our studies of HIV-1 diversity in the East Province found a high proportion of URFs and second generation recombinants (SGRs), suggesting this region of Cameroon may be a breading ground for new CRFs. Herein we present the full-length sequence analysis of one such CRF, composed primarily (66%) of unique, distant lineages of subtypes A and G in alternating regions throughout the genome. This CRF also combines segments in pol and env genes possessing intrasubtype distance (<15%) to the CRF01_AE and CRF02_AG radiations. The genomic composition of this strain comprising gene segments of subtypes A and G as well as CRF01_AE and CRF02_AG defines this strain as a circulating SGR (CSGR), and the 37th CRF to be identified. Furthermore, more than half of CRF19_cpx, a CRF identified in Cuba, clusters with CRF37_cpx, and the clear genetic distance among the viruses in this cluster suggests this strain has been in circulation since the early days of the epidemic. The genetically distant segments comprising CRF37_cpx, which were found to cluster outside the crown groups of previously described viruses, may represent a link to very rare or extinct strains, and, potentially, to understanding the evolutionary history of HIV-1 in this region.

Identification of a novel circulating recombinant form (CRF) 36_cpx in Cameroon that combines two CRFs (01_AE and 02_AG) with ancestral lineages of subtypes A and G.

An array of CRFs have been identified in Cameroon, the most notable being CRF02_AG. HIV-1 in the East Province of Cameroon is particularly diverse: in a recent study, we found a high proportion of unique recombinant forms (URFs). Herein we describe the analysis of the full-length sequences of two of these URFs, which, after preliminary analysis of gag, pol, and env fragments, appeared to be a novel CRF. This novel strain, CRF36_cpx, contains fragments that can be assigned to the CRF01_AE, CRF02_AG, and subtype A and G radiations. Forty percent of the genome can be classified as CRF02_AG, including regions in gag, pol, env, and the accessory genes. Twenty-seven percent is CRF01_AE, comprising the majority of gag, the beginning of env, and the end of env into the 3' LTR. Twenty percent of the genome can be assigned to subtype A, with segments in pol and env. The remaining 13% of the sequence is classifiable as subtype G, in pol and vpu. The subtype A and G lineages formed by the CRF36_cpx sequences are unique and appear ancestral in nature. CRF36_cpx is both the first to combine more than one CRF and the first to include fragments of CRF02_AG. The ancestral sequences present in CRF36_cpx represent a link to extinct strains, and, potentially, insight into the evolution of HIV-1.

Myelinating glia differentiation is regulated by extracellular matrix elasticity.

The mechanical properties of living tissues have a significant impact on cell differentiation, but remain unexplored in the context of myelin formation and repair. In the PNS, the extracellular matrix (ECM) incorporates a basal lamina significantly denser than the loosely organized CNS matrix. Inhibition of non-muscle myosin II (NMII) enhances central but impairs peripheral myelination and NMII has been implicated in cellular responses to changes in the elasticity of the ECM. To directly evaluate whether mechanotransduction plays a role in glial cell differentiation, we cultured Schwann cells (SC) and oligodendrocytes (OL) on matrices of variable elastic modulus, mimicking either their native environment or conditions found in injured tissue. We found that a rigid, lesion-like matrix inhibited branching and differentiation of OL in NMII-dependent manner. By contrast, SC developed normally in both soft and stiffer matrices. Although SC differentiation was not significantly affected by changes in matrix stiffness alone, we found that expression of Krox-20 was potentiated on rigid matrices at high laminin concentration. These findings are relevant to the design of biomaterials to promote healing and regeneration in both CNS and PNS, via transplantation of glial progenitors or the implantation of tissue scaffolds.

Neutralizing antibody patterns and viral escape in HIV-1 non-B subtype chronically infected treatment-naive individuals.

Here we studied the patterns of generation of neutralizing antibodies (NAbs) and virus escape during non-B subtype HIV-1 chronic infection among asymptomatic patients, and established whether a correlation exists between the generation of NAbs and the kinetics of CD4 T-cell decline. Therefore, sequential viruses and plasma obtained at 6 months to one year intervals over a three years period from ten HIV-1 group M subtype A, CRF02_AG, G, and H infected treatment-naïve individuals were tested in neutralization assays. Overall, NAbs were present in all ten individuals, and had the capacity to neutralize autologous virus obtained six months earlier. Eight of the ten subjects showed an increasing capacity to neutralize early viruses and a low capacity to neutralize contemporaneous and later time-point viruses. The neutralizing activities within these individuals resulted in emergence of neutralization resistant viruses, and with the subsequent generation of more NAbs to the emerging resistant viruses. In the remaining two individuals, the capacity to neutralize early, contemporaneous, and later time-point viruses remained conserved. While the kinetics of CD4 T-cell decline varied among all ten individuals, there was no correlation with the capacity to generate NAbs in that, sequential plasmas from individuals with moderately or rapidly declining CD4 T-cells were capable of neutralizing early sequential viruses. We conclude from this study that in non-B subtype chronically infected asymptomatic patients with moderately and rapidly declining CD4 T-cells, potent NAbs are readily generated as the virus evolves to escape the effect of these antibodies.

Defining human immunodeficiency virus (HIV) type 1 immunotypes with six human monoclonal antibodies.

Studies of HIV-1 immunological relatedness have revealed that genetic diversity does not parallel antigenic diversity and have recently shown that HIV-1 strains from different geographic regions from around the world can be grouped into a small number of immunologically defined groups (immunotypes). Previously, the binding patterns of 28 monoclonal antibodies (mAbs) (specific for V3 and C5 of gp120 and cluster I of gp41) with 26 HIV-1 virions obtained globally were determined in a virus binding assay. Analysis of the binding patterns of these 728 mAb/virus combinations now reveals that a particular subset containing six of the 28 mAbs can correctly immunotype 24 of the 26 isolates (92%) into three immunotypes. Like the original panel of mAbs, the subset of six mAbs identified was directed against epitopes in the V3 and C5 regions of gp 120 as well as cluster I of gp41. The binding patterns ("profiles") of these six mAbs with 24 additional HIV-1 virions from Cameroon confirmed that epitopes in V3 and C5 of gp120 and cluster I of gp41 are well exposed on these viruses. Multivariate analysis of the binding patterns of these six mAbs with all 50 viruses (26 obtained globally and 24 obtained from Cameroon) indicates that the viruses from Cameroon have binding profiles similar to viruses from the rest of the world and can be classified into the same three immunotypes that were previously described. This study suggests that a vaccine against HIV-1 need not be based on geographic origin of the virus or on clade, but may better be based on antigenic properties that classify the plethora of different HIV-1 viruses into immunologically defined groups.

Genetic and biological properties of HIV type 1 isolates prevalent in villagers of the Cameroon equatorial rain forests and grass fields: further evidence of broad HIV type 1 genetic diversity.

To understand the evolution of HIV-1, the genetic and biological characteristics of viruses that infect persons living in regions in which the virus has been evolving for several decades must be studied. Thus, we investigated teh genetic subtypes, coreceptor usage, and syncytium-inducing ability of viruses in 47 HIV-1-infected blood samples from individuals living in rural villages in the equatorial rain forest and grass field regions in Cameroon. Heteroduplex mobility analysis (HMA) of gag (part of p24 and p7) and env (C2V5) or sequence and phylogenetic analysis of gag (part of p24 and p7), pol (protease), and env (C2V5), revealed a broad HIV-1 group M genetic diversity. Subtype analysis revealed genetic evidence of seven subtypes (A, C, D, F, G, H, and J) and three circulating recombinant froms (CRFs) (CRF01_AE, CRF02_AG, and CRF11_cpx). Only 15 (32%) of the 47 samples analyzed revealed a concordant subtype in all three genes (gag, pol, and env), while discordant subtypes and CRFs were identified for the remaining 32 (68%) samples. Two patterns of HIV-1 diversity could be discerned in two provinces. While more concordant subtypes in gag, pol, and env genes were identified in villages of South province (10 of 13, 77%), the HIV-1 diversity in the West province was characterized by intersubtype recombinants (63%). Five new intersubtype recombinants were identified including Agag Jpol Genv, Ggag Upol Aenv, AGgag Jpol Aenv, Agag AGpol Henv, and Cgag AGpol AGenv. All of the 40 viruses tested used the R5 coreceptor, of which four also used the X4 coreceptor. Four viruses were able to induce syncytia in MT-2 cells, however, syncytium induction did not correlate with coreceptor usage. This study further reveals the complexity of HIV-1 infection in rural Cameron and points to the future of the global epidemic, which may be characterized by more genetically diverse viruses.

High frequency of HIV-1 dual infections among HIV-positive individuals in Cameroon, West Central Africa.

OBJECTIVES: To determine the frequency of dual inter- and intra-subtype HIV-1 infection among a cohort of 64 longitudinally-studied, HIV-1-positive individuals in Yaoundé, Cameroon. METHODS: Blood was collected every 3-6 months for up to 36 months and RNA was extracted from plasma. Gag fragment (HxB2 location 1577-2040) was amplified by nested RT-PCR, and mixed-time-point Heteroduplex Assays (HDAs) were performed. As heteroduplexes in this assay indicate >or=5% genetic discordance in the gag fragment, their presence reveals dual infection. Results were confirmed by phylogenetic analysis. RESULTS: Heteroduplexes were generated by specimens of 10 subjects (15.6%). Kaplan-Meier nonparametric estimate of maintenance of single infection was calculated; the rate/year of a 2 infection was found to be approximately 11%. Dual infection was identified in the final specimens of five subjects, after as much as 18 months follow-up, while for the remaining five subjects, dual infection was identified in interim specimens within an average of 10 months follow-up. Analysis of samples obtained after dual infection from each of these latter five subjects revealed two patterns: reversion to initial strain, or replacement of initial strain. Four subjects were dually-infected with HIV-1 strains of the same subtype, while 6 were infected with different subtypes. CONCLUSIONS: The high prevalence of recombinant HIV-1 strains in Cameroon may in part be explained by the high frequency of dual infection. In this genetically-diverse HIV-1 milieu, dual infections and the recombinant viruses they generate are strongly driving viral evolution, complicating vaccine strategies.

Neutralization patterns and evolution of sequential HIV type 1 envelope sequences in HIV type 1 subtype B-infected drug-naive individuals.

To design a vaccine that will remain potent against HIV-1, the immunogenic regions in the viral envelope that tend to change as well as those that remain constant over time must be identified. To determine the neutralization profiles of sequential viruses over time and study whether neutralization patterns correlate with sequence evolution, 12 broadly neutralizing plasmas from HIV-1 subtype B-infected individuals were tested for their ability to neutralize sequential primary HIV-1 subtype B viruses from four individuals. Three patterns of neutralization were observed, including a loss of neutralization sensitivity by viruses over time, an increase in neutralization sensitivity by sequential viruses, or a similarity in the sensitivity of sequential viruses to neutralization. Seven to 11 gp160 clones from each sequential virus sample were sequenced and analyzed to identify mutational patterns. Analysis of the envelope sequences of the sequential viruses revealed changes characteristic of the neutralization patterns. Viruses that evolved to become resistant to neutralizing antibodies also evolved with diverse sequences, with most of the changes being due to nonsynonymous mutations occurring in the V1/V2, as well as in the constant regions (C2, C3, C4), the most changes occurring in the C3. Viruses from the patient that evolved to become more sensitive to neutralization exhibited less sequence diversity with fewer nonsynonymous changes that occurred mainly in the V1/V2 region. The V3 region remained constant over time for all the viruses tested. This study demonstrates that as viruses evolve in their host, they either become sensitive or resistant to neutralization by antibodies in heterologous plasma and mutations in different envelope regions account for these changes in their neutralization profiles.

Quasispecies analysis of novel HIV-1 recombinants of subtypes A and G reveals no similarity to the mosaic structure of CRF02_AG.

HIV-1 circulating recombinant form (CRF) 02_AG is responsible for greater than 65% of HIV-1 infections in Cameroon and is widespread across West and West-Central Africa. The parental subtypes A1 and G cocirculate in this part of Africa, and high rates of infection predispose to the generation of AG unique recombinant forms (URFs). Little is known as to whether A1 and G can recombine and thrive in vivo with breakpoints other than those characteristic of CRF02_AG. In this study, six unique recombinant viruses of subtypes A1 and G were identified in two individuals in Cameroon. A 1.5 kb fragment of the reverse transcriptase (RT) region of pol (HXB2 location 2,612-4,159) and the entire env gene (HXB2 location 6,202-9,096) were evaluated by phylogenetic and breakpoint analyses. Each URF was found to have breakpoints different than CRF02_AG, indicating that A and G gene segments are functionally compatible with more than one pattern of recombination. Furthermore, contemporaneous, cultured viruses from these individuals were analyzed, revealing different proportions of URFs compared to those found in plasma, possibly indicating compart mentalization and/or phenotypic variation among the URFs. CRF02_AG emerged from West-Central Africa to become a highly successful viral strain. As such, monitoring the spread of newly emerging AG recombinants is critical not only for understanding the epidemiology of HIV-1, but also in the design of future therapeutics and vaccines appropriate to this part of Africa, and globally.