Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders.

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.

Interdisciplinary patient-centred poststroke care in Follow-up After Stroke, Screening and Treatment (FASST) clinic model: a proof-of-concept pilot study.

Background: Although secondary stroke prevention is important, the optimal outpatient model that improves risk factor control and decreases post-stroke complications effectively has not been established. We created Follow-up After Stroke, Screening and Treatment (FASST), an interdisciplinary clinic involving stroke physicians and pharmacists to address poststroke complications and secondary stroke prevention systemically. We present our approach to assess its proof-of-concept in our pilot study. Methods: We included the patients attending FASST clinic after their hospital discharge. We used validated survey screens to assess for complications: depression, anxiety, sleep disorders, cognitive impairment, disability, social support, quality of life and functional status. Data were collected including risk factors, complication screening results and outcome scores. Clinical pharmacists assessed risk factor control and health-related behaviours for modification. Results: Of the 25 patients enrolled in the interdisciplinary clinic, all had comorbid hyperlipidaemia and hypertension, and 44% had diabetes mellitus. About one-third needed medication changes for risk factor control. On screening, 16% of patients were found to have depression, 12% had anxiety and 20% had sleep apnoea. These patients were either managed in the clinic or were referred to relevant subspeciality clinics. The status of risk factor control was assessed in all patients, and 32% had medications adjustments. Conclusion: Our preliminary data found that FASST clinic model is feasible and potentially useful. It represents an integrated approach to post-stroke care, with pharmacist collaboration to improve risk factor control, while assessing for poststroke complications. Further study is needed to improve health outcomes through integrated poststroke care.

A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells.

Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of betaIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells.

BACKGROUND: Although extensive research has been performed to control differentiation of neural stem cells - still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation - allowing for an increase in percentage yield of neuronal cells. RESULTS: Uncommitted GFAP and SOX2 positive neural progenitors - so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100beta-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells--i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. CONCLUSION: We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells - such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

Synucleinopathies in neurodegenerative diseases: Accomplices, an inside job and selective vulnerability.

Pathogenesis of degenerative diseases is complex and multifaceted. The disease phenotypes depend on the location of injury/damage in the brain networks and pathologically are characterized by loss of brain cells. The reason for this loss appears to be an accumulation of misfolded and dysfunctional proteins that trigger apoptotic cell death. The role of alpha-synuclein mutations, its interaction with other proteins and the cellular environment is discussed in the context of selective neuron loss.

Neuronal and astrocytic cells, obtained after differentiation of human neural GFAP-positive progenitors, present heterogeneous expression of PrPc.

PrP(c) is a cellular isoform of the prion protein with an unknown normal function. One of the putative physiological roles of this protein is its involvement in cell differentiation. Recently, in vitro and in vivo studies showed that GFAP-positive cells have characteristics of stem/progenitor cells that generate neurons and glia. We used an in vitro model of human neurogenesis from GFAP-positive progenitor cells to study the expression of PrP(c) during neural differentiation. Semi-quantitative multiplex-PCR assay and Western blot analysis revealed a significant increase of PRNP expression level in differentiated cells compared to undifferentiated cell population. As determined by immunocytochemistry followed by a quantitative image analysis, the PrP(c) level increased significantly in neuronal cells and did not increase significantly in glial cells. Of note, glial and neuronal cells showed a very large heterogeneity of PrP(c) expression. Our results provide the basis for studying the role of PrP(c) in cell differentiation and neurogenesis from human GFAP-positive progenitor cells.

Pyramidal and extrapyramidal dysfunction as a sequela of hypoxic injury: case report.

BACKGROUND: The clinical and radiological aspects of hypoxic brain injury without ischemia are not well characterized. A spectrum of clinical manifestations have been observed in patients that recover from hypoxic brain injury, including a subset that demonstrate persistent motor system disturbances. Early Magnetic Resonance Imaging (MRI) studies have shown abnormalities in basal ganglia, cerebral and cerebellar cortex. CASE PRESENTATION: A 23-year-old man was affected by acute respiratory failure after drug overdose. His clinical condition progressed from coma to partial recovery with persistent lack of control and stiffness in the lower extremities. MRI of the brain showed evolving lesions in the cerebellum, globus pallidus and motor cortex that correlated with neurological signs. CONCLUSION: A careful analysis of this case and a review of the relevant literature indicate that the clinical residua after recovery from hypoxic injury to the brain is predominantly disorders of the motor system, and the MRI manifestations as well as the clinical presentation can evolve over time. Understanding more of the factors that affect hypoxic brain injury can be helpful in determining the clinical outcome and management of these patients.

The spectrum of neuromyelitis optica: a case of NMO with extensive brain stem involvement.

Neuromyelitis optica (NMO), regarded as a distinct clinical entity from multiple sclerosis (MS), is generally characterized by demyelinating lesions involving optic nerves and spinal cord with sparing of the brain. We report a case with initial clinical, radiological and serological features consistent with NMO, but with concomitant extensively clinical and radiological involvement of the brain stem. Although there are well defined criteria for NMO that restricts the lesions to optic nerves and spinal cord, the case presented here and the review of literature support the idea that NMO may present as a spectrum of clinical and radiological entities rather than the confining clinical criteria that has been hitherto applied.

Intracerebral hemorrhage with intraventricular extension and no hydrocephalus may not increase mortality or severe disability.

This paper aimed to test the hypothesis that intraventricular extension of spontaneous intracerebral hemorrhage (ICH) in the absence of hydrocephalus is not associated with increased mortality or severe disability. We performed a retrospective consecutive cohort study of patients with primary spontaneous ICH who were admitted to a single institution. Multivariate logistic regression analysis was used to assess the association of each variable with functional outcome as measured by the modified Rankin Scale (mRS). A total of 164 patients met our inclusion criteria and were included in the study. Only hydrocephalus (p=0.002) and hematoma volume (p=0.006) were significantly associated with mortality or poor functional outcome (mRS of 3 to 6). In contrast, the presence of intraventricular hematoma was not independently associated with poor functional outcome. The presence of intraventricular extension of ICH in the absence of hydrocephalus may not increase mortality or disability.

"I think therefore I am": new prospects for neural prostheses.

Three categories of neuro-prosthetics are being developed and used in increasing frequency to ameliorate neurologic disability. They are sensory receivers, motor effectors and brain 'stimulators'. The interfaces necessary to drive these devices are critical for their functioning. The following is a brief commentary on brain machine interface.

Derivation of neuronal cells from fetal normal human astrocytes (NHA).

Pathological changes in most neurological diseases are marked by cell loss. To understand the mechanisms of neurogenesis and brain repair at a cellular level, observation on less complex systems provide valuable knowledge which offer the basis for therapeutic interventions. This has been the impetus for neural cell culture studies and the development of in vitro models. Here, we provide protocols for differentiation into neuronal lineage of commercially available normal human astrocytes (NHA) that are isolated from normal fetal human brain (Lonza, Inc.). It is known that some of GFAP positive astrocytic cells have stem/progenitor cell characteristics; however, understanding of the human GFAP positive cells with these characteristics remains limited. The genesis of neuronal lineage cells from the NHA occurs in adherent culture conditions by removal of serum and exposure to bFGF. When transferred to serum-free medium supplemented with bFGF, NHA cells generate neuronal precursors that express doublecortin, nestin and are negative for GFAP. After withdrawal of bFGF they mature into neurons. The average time required for generation of neuronal cells using this protocol is about 3 weeks. Our model of neurogenesis captures a contained in vitro system consisting of both neurons and glia. This "human brain in a dish" model can be used to assay the effects of interventions on developing human neurons at a cellular and molecular level and is also suitable for modeling of various aspects of human diseases and testing of novel therapeutic strategies.

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.

Of sleep, seizures and networks.

A common property of sleep and epileptic seizures is that both occur because of alteration in network activity within the nervous system. This is represented by an oscillating synchronization of spiking in large groups of neurons from the ever evanescent chaotic yet coherent patterns of activity that underlie sensory and motor operations of the brain. The idea that both slow wave sleep and epileptic seizures may have similar functions in brain homeostasis by purging and consolidating environmental cues is discussed. TMS can be used as a tool to investigate these patterns.

Bone marrow-derived mesenchymal stem cells undergo JCV T-antigen mediated transformation and generate tumors with neuroectodermal characteristics.

There is now accumulating evidence showing that some tumors may arise from transformed stem cells. In this study we demonstrate that adult bone marrow- derived mesenchymal stem cells (MSCs) undergo neoplastic transformation induced by the human polyomavirus JCV, early protein, T-antigen, and are tumorigenic when transplanted into the flanks of Nude mice as compared to non-transformed MSCs. Histologically, the tumors are heterogeneous with mesenchymal and neural crest characteristics as evidenced by expression of the neural crest markers p75, SOX-10, and S-100, with populations of tumor cells exhibiting characteristics of primitive neuroectodermal cells. In addition, a subset of T-antigen positive tumor cells exhibit a high proliferation index as detected by Ki-67 labeling, and co-express CD133, a marker which is expressed on cancer stem cells. These results show that tumors with neuroectodermal characteristics may arise from transformation of MSCs, a globally accessible adult stem cell with multipotent differentiation capacity. In light of earlier reports on the association of JCV with a broad variety of human tumors, our data suggests that T-antigen transformation of adult stem cells with a multipotent capacity can serve as a possible common origin for some of these cancers, and offers a novel model for oncogenesis.

Brain to music to brain!

It has been implicitly understood that culture and music as collective products of human brain in turn influence the brain itself. Now, imaging and anatomical data add substance to this notion. The impact of playing piano on the brain of musicians and its possible effects on cultural and neurological evolution are briefly discussed.

A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages.

Glial fibrillary acidic protein (GFAP)-positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP-positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of betaIII-tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) betaIII-tubulin- and nestin-positive cells and (ii) GFAP- and nestin-positive cells. These cells, when subsequently cultured in serum-free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down-regulated and betaIII-tubulin expression became robust. Cells of glial lineage differentiated by down-regulating nestin expression and up-regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.

Human fibroblast-derived cell lines have characteristics of embryonic stem cells and cells of neuro-ectodermal origin.

Fibroblasts are the most ubiquitous cells in complex organisms. They are the main cells of stromal tissue and play an important role in repair and healing of damaged organs. Here we report new data-initially serendipitous findings-that fibroblast-derived cell line (human fetal lung derived cells, MRC-5) have the morphology, growth rate and gene expression pattern characteristic of embryonic stem cells and cells of neuro-ectodermal origin. We have developed a serum-free culture system to maintain these cells in proliferative state. We discovered that, at proliferative state, these cells express transcription factors of pluripotent cells, OCT-3/4 and REX-1, and embryonic cell surface antigens SSEA-1, SSEA-3, and SSEA-4, as well as TRA-1-60 and TRA-1-81. In addition to embryonic cell markers, the fibroblasts expressed neuroectodermal genes: Musashi-1, nestin, medium neurofilament, and beta-III tubulin. RT-PCR data revealed that mesencephalic transcription factors, Nurr-1 and PTX-3, were also expressed in MRC-5 cells, and that these cells could be induced to express tyrosine hydroxylase (TH). Expression of TH followed down-regulation of genes associated with cell proliferation, OCT-3/4, REX-1, and beta-catenin. These data indicate that the cells commonly known as fibroblasts have some of the characteristics of stem cells, and can be induced to become neuroectodermal cells and perhaps even mature neurons.