Miso Mitkovski

Satb2 Is a Postmitotic Determinant for Upper-Layer Neuron Specification in the Neocortex

Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.

Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images

Methods of blind source separation are used in many contexts to separate composite data sets according to their sources. Multiply labeled fluorescence microscopy images represent such sets, in which the sources are the individual labels. Their distributions are the quantities of interest and have to be extracted from the images. This is often challenging, since the recorded emission spectra of fluorescent dyes are environment- and instrument-specific. We have developed a nonnegative matrix factorization (NMF) algorithm to detect and separate spectrally distinct components of multiply labeled fluorescence images. It operates on spectrally resolved images and delivers both the emission spectra of the identified components and images of their abundance. We tested the proposed method using biological samples labeled with up to four spectrally overlapping fluorescent labels. In most cases, NMF accurately decomposed the images into contributions of individual dyes. However, the solutions are not unique when spectra overlap strongly or when images are diffuse in their structure. To arrive at satisfactory results in such cases, we extended NMF to incorporate preexisting qualitative knowledge about spectra and label distributions. We show how data acquired through excitations at two or three different wavelengths can be integrated and that multiple excitations greatly facilitate the decomposition. By allowing reliable decomposition in cases where the spectra of the individual labels are not known or are known only inaccurately, the proposed algorithms greatly extend the range of questions that can be addressed with quantitative microscopy.

Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system

During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.

The SNARE protein SNAP-29 interacts with the GTPase Rab3A: Implications for membrane trafficking in myelinating glia.

During myelin formation, vast amounts of specialized membrane proteins and lipids are trafficked toward the growing sheath in cell surface‐directed transport vesicles. Soluble N‐ethylmaleimide‐sensitive factor (NSF) attachment proteins (SNAPs) are important components of molecular complexes required for membrane fusion. We have analyzed the expression profile and molecular interactions of SNAP‐29 in the nervous system. In addition to its known enrichment in neuronal synapses, SNAP‐29 is abundant in oligodendrocytes during myelination and in noncompact myelin of the peripheral nervous system. By yeast two‐hybrid screen and coimmunoprecipitation, we found that the GTPases Rab3A, Rab24, and septin 4 bind to the N‐terminal domain of SNAP‐29. The interaction with Rab24 or septin 4 was GTP independent. In contrast, interaction between SNAP‐29 and Rab3A was GTP dependent, and colocalization was extensive both in synapses and in myelinating glia. In HEK293 cells, cytoplasmic SNAP‐29 pools were redistributed upon coexpression with Rab3A, and surface‐directed trafficking of myelin proteolipid protein was enhanced by overexpression of SNAP‐29 and Rab3A. Interestingly, the abundance of SNAP‐29 in sciatic nerves was increased during remyelination and in a rat model of Charcot‐Marie‐Tooth disease, two pathological situations with increased myelin membrane biogenesis. We suggest that Rab3A may regulate SNAP‐29‐mediated membrane fusion during myelination.

Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinsons disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinsons disease.

Doxycycline restrains glia and confers neuroprotection in a 6‐OHDA Parkinson model

Neuron–glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinsons disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinsons disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6‐OHDA mouse model of Parkinsons disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6‐OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6‐OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinsons disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinsons disease and other neurodegenerative diseases linked to glia function.

Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.

Modulation of dopamine uptake by nitric oxide in cultured mesencephalic neurons

Strong evidence obtained from in vivo and ex-vivo studies suggests the existence of interaction between dopaminergic and nitrergic systems. Some of the observations suggest a possible implication of nitric oxide (NO) in dopamine (DA) uptake mechanism. The present work investigated the interaction between both systems by examining the effect of an NO donor, sodium nitroprusside (SNP), associated with the indirect DA agonist, amphetamine (AMPH) on tritiated DA uptake in cultures of embryonic mesencephalic neurons. Consistent with the literature, both AMPH (1, 3 and 10 microM) and SNP (300 microM and 1 mM) inhibited DA uptake in a dose-dependent manner. In addition, the inhibition of DA uptake by AMPH (1 and 3 microM) was significantly increased by the previous addition of SNP (300 microM). The implication of NO in this interaction was supported by the fact that the free radical scavenger N-acetyl-L-Cysteine (500 microM) significantly increased DA uptake and completely abolished the effect of SNP, leaving unaffected that from AMPH on DA uptake. Further, double-labeling immunohistochemistry showed the presence of tyrosine hydroxylase- (TH, marker for dopaminergic neurons) and neuronal NO synthase- (nNOS, marker for NO containing neurons) expressing neurons in mesencephalic cultures. Some dopaminergic neurons also express nNOS giving further support for a pre-synaptic interaction between both systems. This is the first work demonstrating in mesencephalic cultured neurons a combined effect of an NO donor and an indirect DA agonist on specific DA uptake.

Axonal degeneration in an Alzheimer mouse model is PS1 gene dose dependent and linked to intraneuronal Aβ accumulation

Abnormalities and impairments in axonal transport are suggested to strongly contribute to the pathological alterations underlying AD. The exact mechanisms leading to axonopathy are currently unclear, but it was recently suggested that APP expression itself triggers axonal degeneration. We used APP transgenic mice and crossed them on a hemi- or homozygous PS1 knock-in background (APP/PS1KI). Depending on the mutant PS1 dosage, we demonstrate a clear aggravation in both plaque-associated and plaque-distant axonal degeneration, despite of an unchanged APP expression level. Amyloid-β (Aβ) peptides were found to accumulate in axonal swellings as well as in axons and apical dendrites proximate to neurons accumulating intraneuronal Aβ in their cell bodies. This suggests that Aβ can be transported within neurites thereby contributing to axonal deficits. In addition, diffuse extracellular Aβ deposits were observed in the close vicinity of axonal spheroids accumulating intracellular Aβ, which might be indicative of a local Aβ release from sites of axonal damage.

Investigations into Potential Extrasynaptic Communication between the Dopaminergic and Nitrergic Systems

Nitric oxide is unconstrained by cell membranes and can therefore act along a broad distance as a volume transmitter. Spillover of nitric oxide between neurons may have a major impact on central nervous system diseases and particularly on neurodegeneration. There is evidence whereby communication between nitrergic and dopaminergic systems plays an essential role in the control of the nigrostriatal pathway. However, there is sparse information for either the coexistence or overlap of nitric oxide and dopaminergic structures. The dual localization of immunoreactivity for nitric oxide synthase (NOS) and tyrosine hydroxylase, enzymes responsible for the synthesis of nitric oxide and dopamine, respectively, was examined in neurons of the nigrostriatal pathway in the rat brain by means of a double-immunohistochemical method and confocal laser scanning microscopy, acquired at the resolution limit. After perfusional fixation, the brains were cut and double-immunostained. A proximity analysis of tyrosine hydroxylase and NOS structures was done using binary masks generated from the respective maximum projections, using confocal laser microscopy. Unrevealed regions were determined somatodendritic positive for both NOS and tyrosine hydroxylase, within an image limit resolution at 2 μm-wide margin. The described interconnected localization of nNOS(+) and TH(+) containing neuronal fibers and cells bodies in the nigrostriatal pathway propose a close anatomical link between the two neurotransmitters.