Mutations in the gene encoding alpha-synuclein (asyn) causes autosomal-dominant, in the parkin gene autosomal-recessive forms of Parkinson's disease (PD). The pathophysiology of PD is poorly understood, even though published evidence suggests a role for mitochondria in the pathogenesis. To gain insight into the influence of asyn and parkin on mitochondrial integrity and function, we have generated several mono-mutant mouse lines expressing doubly mutated human asyn (hm(2)asyn) under the control of two different promoters, or a targeted deletion of Parkin (Parkin-Exon3-knockout). Both mouse lines were crossed to generate the double-mutant. Here we compare the ultrastructure and functional properties of mitochondria in the substantia nigra (SN), the striatum, the cerebral cortex (Cx) and skeletal muscle of young (2-3 months) and aged (12-14 months) mono- and double-mutants mice. We observed severe genotype-, age- and region-dependent morphological alterations of mitochondria in neuronal somata. The number of structurally altered mitochondria was significantly increased in the SN of both double-mutants and in the Cx of one mono- and one double-mutant line. These alterations coincided with a reduced complex I capacity in the SN, but were neither accompanied by alterations in the number or the size of the mitochondria nor by leakage of cytochrome c, Smac/DIABLO or Omi/HtrA2. None of the transgenic animals developed any gross histopathological abnormalities or overt motor disabilities. Together our results provide compelling evidence that (i) both, asyn and parkin are relevant for mitochondrial integrity, (ii) the influence of these proteins on mitochondria are age- and tissue-specific and (iii) changes of mitochondrial morphology do not inevitably cause functional impairments.

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function. Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia. The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. The removal of the desialylated neurites was mediated via the complement receptor-3 (CR3; CD11b/CD18). Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures. In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: (i) cultivation of ES cells, (ii) formation and differentiation of embryoid bodies, (iii) differentiation into neuroectodermal lineage and isolation of myeloid precursor cells, (iv) differentiation into microglial precursor cells and (v) cultivation of ES cell-derived microglial precursors (ESdMs). The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia. Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

KEY WORDS microglia; immunoreceptor tyrosine-based activation motif; immunoreceptor tyrosine-based inhibition motif; triggering receptor expressed on myeloid cells-2; complement receptor-3; DAP12; sialic acid-binding immunoglobulin superfamily lectins; sialic acid ABSTRACT Microglia sense intact or lesioned cells of the central nervous system (CNS) and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors. Fc receptors and DAP12 (TYROBP)-associated receptors such as microglial triggering receptor expressed on myeloid cells-2 (TREM2) and the complement receptor-3 (CR3, CD11b/CD18) trigger the immunoreceptor tyrosine-based activation motif (ITAM)-signaling cascade, resulting in microglial activation, migration, and phagocytosis. Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif (ITIM)-signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis. In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM. Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM-or ITAM-signaling cascade accordingly. V V C 2012 Wiley Periodicals, Inc.

We recently described mitochondrial pathology in neurons of transgenic mice with genes associated with Parkinson's disease (PD). Now we describe severe mitochondrial damage in glial cells of the mesencephalon in mice carrying a targeted deletion of parkin (PaKO) or overexpressing doubly mutated human alpha-synuclein (asyn). The number of mitochondria with altered morphology in glial cells is cell type-dependent, but always higher than in neurons. Interestingly, mitochondrial damage also occurs in mesencephalic glia of mice carrying mutated asyn controlled by the tyrosine hydroxylase promoter. Such mice do not show glial expression of the transgene, but show expression in neighboring neurons. However, we found strong overexpression of endogenous asyn in mesencephalic astrocytes from these mice. Cortical astrocytes neither display enhanced asyn expression nor mitochondrial damage. Cultivated mesencephalic astrocytes from newborn transgenic mice display various functional defects along with the morphological damage of mitochondria. First, the mitochondrial Ca(2+)-storage capacity is reduced in asyn transgenic mesencephalic astrocytes, but not in astrocytes from PaKO. Second, the expression of the mitochondrial protein PTEN-induced putative kinase is constitutively increased in asyn transgenic mice, while in PaKO it reacts to oxidative stress by overexpressing this protein along with other mitochondria-related proteins. Third, the neurotrophic effects exerted by control astrocytes, stimulating cortical neurons from healthy mice to develop longer processes and larger neuronal areas, are lacking in co-cultures with transgenic mesencephalic astrocytes. In summary, glial mitochondria from transgenic mice display morphological and functional alterations. Such transgenic astrocytes fail to influence neuronal differentiation, reflecting an important role that glia may play in PD pathogenesis.