Peter C. Mabie

Bone morphogenetic proteins in the nervous system

Bone morphogenetic proteins (BMPs) are a rapidly expanding subclass of the transforming growth factor superfamily. BMP ligands and receptor subunits are present throughout neural development within discrete regions of the embryonic brain and within neural crest-derived pre- and post-migratory zones. BMPs initially inhibit the formation of neuroectoderm during gastrulation while, within the neural tube, they act as gradient morphogens to promote the differentiation of dorsal cell types and intermediate cell types throughout co-operative signaling. In the peripheral nervous system, BMPs act as instructive signals for neuronal lineage commitment and promote graded stages of neuronal differentiation. By contrast, within the CNS, these same factors promote astroglial lineage elaboration from embryonic subventricular zone progenitor cells, with concurrent suppression of the neuronal or oligodendroglial lineages, or both. In addition, BMPs act on more lineage-restricted embryonic CNS progenitor cells to promote regional neuronal survival and cellular differentiation. Furthermore, these versatile cytokines induce selective apoptosis of discrete rhombencephalic neural crest-associated cellular populations. These observations suggest that the BMPs exhibit a broad range of cellular and context-specific effects during multiple stages of neural development.

Developmental changes in progenitor cell responsiveness to cytokines

Multipotent progenitor cells have been identified within periventricular generative zones of the developing and adult brain. To determine whether the environmental responsiveness of these cells changes during development, progenitor cells were cultured from embryonic, postnatal, and adult rat brain in the presence of either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Embryonic cells cultured as intact progenitor neurospheres proliferated more robustly in response to bFGF than to EGF, whereas proliferation of postnatal and adult progenitor cells was enhanced more by EGF than bFGF. Progenitor cells generated in the presence of either bFGF or EGF had the capacity to generate neurons, astrocytes, and oligodendrocytes at all developmental stages. Most embryonic and neonatal bFGF-generated cells differentiated predominantly into neurons, whereas late stage embryonic and neonatal EGF-generated progenitors largely remained in an undifferentiated state. However, later postnatal and adult progenitor species, irrespective of whether they were generated in the presence of bFGF or EGF, gave rise preferentially to astrocytes. Treatment with bone morphogenetic protein (BMP)2 or BMP7 enhanced astroglial differentiation and suppressed oligodendroglial differentiation of both EGF- and bFGF-generated progenitor species, suggesting that the effects of the BMPs are not dependent on EGF receptor activation. Thus, while central nervous system (CNS) progenitor cells retain multipotent capacity and responsiveness to the BMPs throughout development, they exhibit significant changes in other cellular response properties, perhaps reflecting differences in the requirements for specific generative versus regenerative events.

Cytokines regulate the cellular phenotype of developing neural lineage species

The patterns and mechanisms of action of inductive signals that orchestrate neural lineage commitment and differentiation in the mammalian brain are incompletely understood. To examine these developmental issues, we have utilized several culture systems including conditionally immortalized cell lines, subventricular zone progenitor cells and primary neuronal cultures. A neural stem and progenitor cell line (MK31) was established from murine embryonic hippocampus by retroviral transduction of temperature‐sensitive alleles of the simian virus 40 large tumor antigen. At the non‐permissive temperature for antigen expression (39°C) in serum‐free media, the neural stem cells give rise to a series of increasingly mature neuronal progenitor and differentiated cellular forms under the influence of a subset of hematolymphopoietic cytokines (interleukins 5, 7, 9 and 11), when individually co‐applied with transforming growth factor α, after pretreatment with basic fibroblast growth factor. These cellular forms elaborated a series of progressively more mature neurofilament proteins, a sequential pattern of ligand‐gated channels, and inward currents and generation of action potentials with mature physiological properties. Because the factors regulating the development of central nervous system astrocytes have been so difficult to define, we have chosen to focus, in this manuscript, on the elaboration of this cell type. At 39°C, application of a subfamily of bone morphogenetic proteins of the transforming growth factor β superfamily of growth factors sanctioned the selective expression of astrocytic progenitor cells and mature astrocytes, as defined by sequential elaboration of the Yb subunit of glutathione‐S‐transferase and glial fibrillary acidic protein. These lineage‐specific cytokine inductive relationships were verified using subventricular zone neural progenitor cells generated by the application of epidermal growth factor, alone or in combination with basic fibroblast growth factor, to dissociated cellular cultures derived from early embryonic murine brain, a normal non‐transformed developmental population. Finally, application of a different series of cytokines from five distinct factor classes (basic fibroblast growth factor, platelet‐derived growth factor‐AA, insulin‐like growth factor 1, neurotrophin 3 and representative gp130 receptor subunit‐related ligands) caused the elaboration of oligodendroglial progenitor species and post‐mitotic oligodendrocytes, defined by progressive morphological maturation and the expression of increasingly adbanced oligodendroglial and oligodendrocyte lineage markers. In addition, seven different gp130‐associated neuropoietic (ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin‐M) and hematopoietic (interleukins 6, 11, 12, granulocyte‐colony stimulating factor) cytokines exhibited differential trophic effects on oligodendroglial lineage maturation and factor class interactions. Examination of the expression of hematolymphopoietic cytokines and their receptors in brain and neural cultures has confirmed that these epigenetic signals are present at the appropriate development times to mediate their neurotrophic actions. These cytokines signal through alternate receptor subunit motifs distinct from those of the traditional neurotrophins. The bone morphogenetic protein ligand, in particular, exhibit a complex spatiotemporal pattern of transcript expression that suggests a broad spectrum of developmental roles for these transforming growth factor β subclass factors.

Developmental changes in neural progenitor cell lineage commitment do not depend on epidermal growth factor receptor signaling

Multipotent neural progenitor cells become progressively more biased towards a glial fate during development coincident with an increase in expression of the epidermal growth factor receptor (EGFR). To determine whether differences in lineage commitment of neural progenitor cells from different stages are causally related to expression of the EGFR and whether generation of glia is EGFR‐dependent, we used an EGFR‐specific tyrosine kinase inhibitor, PD158780, to block the activation of EGFR in progenitor cells. Treatment of cultured neonatal progenitor cells with PD158780 completely blocked EGF‐induced proliferation of the cells but did not affect bFGF‐induced proliferation. Nevertheless, treatment with the inhibitor failed to inhibit the generation of astroglia in the presence of either EGF or bFGF. Treatment with bone morphogenetic protein‐2 (BMP2) enhanced astroglial differentiation and suppressed oligodendroglial (OL) differentiation. PD158780 treatment had no effect on the BMP2‐induced astroglial differentiation or OL suppression. These observations suggest that the generation of astroglia is not dependent on EGFR activation. Because it was still possible that the progenitor cell responses reflected a prior history of EGFR signaling, rat forebrain cells were cultured in the presence of PD158780 from a time (E12.5) preceding expression of the EGFR. After time in culture, the E12.5 cells expressed EGFR by Western analysis both in the presence and in the absence of PD158780, but activation of EGFR kinase (receptor autophosphorylation) was undetectable in the presence of PD158780 and the cells did not proliferate in response to EGF. Nevertheless, astroglial differentiation was normal in PD158780‐treated cells both in the absence and in the presence of BMPs or CNTF. Furthermore, the propensity towards glial differentiation increased with time in culture even in the absence of EGFR signaling. This suggests that the increased bias towards glial differentiation during development does not depend on EGFR signaling. J. Neurosci. Res. 59:312–320, 2000

Microglial lineage species are expressed in mammalian epidermal growth factor-generated embryonic neurospheres

The epigenetic signals and progenitor cell species involved in progressive neural maturation in the mammalian brain are poorly understood. Although these complex developmental issues can be examined in cultures of generative zone progenitor cells, analysis of signaling relationships in complex progenitor cell systems requires the meticulous definition of the cellular complement at each developmental stage. The presence of microglia within the generative zone cultures would further complicate these developmental analyses. Utilizing the microglial markers Griffonia simplicifolia B4 isolectin, carbocyanine dye‐acetylated low density lipoprotein, F4/80, and Mac‐1 we now report the presence of microglia within cultures of late embryonic murine epidermal growth factor‐derived generative zone progenitor cells. Cytokine treatment of serially passaged epidermal growth factor‐generated neurospheres altered the phenotype of the microglia in culture. Macrophage colony‐stimulating factor treatment promoted the expression of spindle‐shaped microglia, whereas granulocyte‐macrophage colony‐stimulating factor treatment promoted the elaboration of flat and amoeboid microglia. Treatment with microglial‐conditioned medium or 10% non‐heat inactivated fetal calf serum led to an increased complement of both phenotypes. Microglia could be generated from single isolated neurospheres, and there were differences in the number of microglial lineage species obtained from distinct oligopotent progenitor cells, raising the possibility that a complement of this cellular lineage may be derived from a progenitor cell present within the generative zones. These observations indicate that microglia are present within the generative zone progenitor cell system, and this system thus represents an important experimental resource to examine the progenitor cell maturation and the origin of the microglial lineage.

Paracrine regulation of colony-stimulating factor-1 in medulloblastoma: Implications for pathogenesis and therapeutic interventions

OBJECTIVE Colony-stimulating factor (CSF)-1, a chemotactic and mitogenic factor for macrophages and microglia, is expressed in a variety of nervous system tumors and when present in nonneural malignancies, is associated with marked inflammatory infiltrates, dissemination, and poorer prognosis. This study investigated the paracrine effects of CSF-1 production by medulloblastoma cells on the macrophage/microglial lineage. METHODS A recurrent metastatic desmoplastic medulloblastoma was isolated from a 26-year-old man and propagated in tissue culture. Cellular phenotype and proliferation were assessed by immunocytochemical techniques; transcript expression for CSF-1, granulocyte macrophage-CSF, interleukin-3, and c-fms (the receptor for CSF-1) was examined with reverse transcriptase-polymerase chain reaction; and conditioned media and coculture paradigms were used to study cytokine effects on cellular proliferation. RESULTS Serially passaged cells were uniformly immunoreactive for two lineage-independent neuroepithelial markers, nestin and vimentin. A subpopulation of cells with morphological characteristics of early differentiation stained for neurofilament 66 (7%) and microtubule-associated protein (5%) (markers of early neuronal precursors and postmitotic neurons, respectively) and for the Yp subunit of glutathione-S-transferase (3%) (a marker of early oligodendroglial progenitors). Tumor cells expressed transcripts for CSF-1, but not for granulocyte macrophage-CSF, interleukin-3, or c-fms. Treatment of microglia with serum-free medulloblastoma-conditioned media significantly increased proliferation (P < 0.001), suggesting the secretion of CSF-1. Coculture of medulloblastoma cells and microglia significantly increased proliferation of both cell types (each condition, P < 0.01). CONCLUSION These observations suggest that CSF-1 mediates important paracrine interactions between transformed cells and the immune system, resulting in increased growth rate and metastatic potential. Future therapeutic goals need to include immunotherapeutic protocols to modulate this interaction.

Two-stage screening for early dementia in primary care

ABSTRACT Objective: The objective was to compare two screening strategies for dementia in an urban primary care clinic, serving a low-education, minority community composed largely of Latino and African American patients. Method: Two hundred and fifty-seven patients underwent two-stage patient-based screening (PBS) and informant-based screening (IBS) followed by a diagnostic evaluation. In the first stage, PBS included brief tests of episodic memory (Memory Impairment Screen), semantic memory (Animal Fluency), and executive function (Reciting Months Backwards). For IBS, the first stage consisted of the short Informant Questionnaire on Cognitive Decline in the Elderly, administered to a family member or friend. Patients who screened positive in the first stage of either strategy underwent testing with the picture version of the Free and Cued Selective Reminding Test with Immediate Recall to identify memory impairment. Sensitivity, specificity, and positive and negative predictive values were computed for various cutoffs of each test and combination of tests. Dementia was diagnosed using Diagnostic and Statistical Manual of Mental Disorders–Fourth Edition (DSM–IV) criteria without access to the screening test results. Results: We identified 66 patients (25.7%) with previously undiagnosed dementia. Sensitivity was the same (77%) for both strategies but specificity was higher for IBS than for PBS (92% versus 83%). IBS’s higher specificity makes it the preferred strategy if a knowledgeable informant is available. Conclusion: Unrecognized dementia is common in primary care. Case-finding can be improved using either PBS or IBS two-stage screening strategies.

Identifying memory impairment and early dementia in primary care

This study examined the operating characteristics of two‐stage case finding to identify memory impairment and very mild dementia.

Corticosteroid-resistant bulbar neurosarcoidosis responsive to intravenous immunoglobulin

We report an intriguing case of corticosteroid-resistant bulbar neurosarcoidosis responding to intravenous immunoglobulin. A 37-year-old man presented with dysphagia to solids and liquids, dysphonia, fatigue and 50 lb weight loss over 2 months. We suspected sarcoidosis, based on an elevated serum angiotensin-converting enzyme concentration and hilar lymphadenopathy on chest imaging; we subsequently confirmed this after transbronchial biopsy found non-caseating granulomas. MR scan of brain was normal; barium swallow showed severe oropharyngeal dysphagia and electromyography identified bulbar muscle denervation. He took corticosteroids for 3 weeks without improvement, requiring a percutaneous endoscopic gastrostomy tube for nutrition, but then he promptly improved with a 2-day course of intravenous immunoglobulin. Although there have been a few reports of intravenous immunoglobulin helping peripheral neurosarcoidosis, this case suggests that it also helps bulbar neurosarcoidosis. This case shows that bulbar neurosarcoidosis can mimic the clinical and electrophysiological features of fatal neurological disorders such as progressive bulbar palsy. The case illustrates the diagnostic challenge particularly when imaging is inconclusive and there is no response to corticosteroids. It also suggests that intravenous immunoglobulin can be considered before cytotoxic therapy for corticosteroid-resistant neurosarcoidosis, particularly in decompensated patients, given its favourable side effect profile. We also review the literature on bulbar neurosarcoidosis.

Anosognosia for right hemiplegia from dominant anterior cerebral artery stroke.

ACKNOWLEDGMENTS We would like to thank Dr. Sheva Mann for her help in editing the letter, Dr. Lisa Deutsch, for statistical analysis, and Professor Josef Wisman for the vitamin D determinations. Conflict of Interest: The editor in chief has reviewed the conflict of interest checklist provided by the authors and has determined that the authors have no financial or any other kind of personal conflict with this letter. Author Contributions: All of the authors took part in every aspect of this letter including data analysis and manuscript preparation. Sponsor’s Role: No sponsors.