Enrica Boda

Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28

Autosomal dominant spinocerebellar ataxias (SCAs) are genetically heterogeneous neurological disorders characterized by cerebellar dysfunction mostly due to Purkinje cell degeneration. Here we show that AFG3L2 mutations cause SCA type 28. Along with paraplegin, which causes recessive spastic paraplegia, AFG3L2 is a component of the conserved m-AAA metalloprotease complex involved in the maintenance of the mitochondrial proteome. We identified heterozygous missense mutations in five unrelated SCA families and found that AFG3L2 is highly and selectively expressed in human cerebellar Purkinje cells. m-AAA–deficient yeast cells expressing human mutated AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment and deficiency of respiratory chain complex IV. Structure homology modeling indicates that the mutations may affect AFG3L2 substrate handling. This work identifies AFG3L2 as a novel cause of dominant neurodegenerative disease and indicates a previously unknown role for this component of the mitochondrial protein quality control machinery in protecting the human cerebellum against neurodegeneration.

Selection of Reference Genes for Quantitative Real-time RT-PCR Studies in Mouse Brain

Since a growing number of studies based on the real-time reverse transcriptase polymerase chain reaction (RT-PCR) continue to be published in order to highlight genes specifically involved in brain development, maturation, and function, the identification of reference genes suitable for this kind of experiments is now an urgent need in the neuroscience field. The aim of this work was to verify the suitability of some very common housekeeping genes (such as Gapdh, 18s, and B2m) and of some relatively new control genes (such as Pgk1, Tfrc, and Gusb) during mouse brain maturation. We tested the candidate reference genes in mouse whole brain, cerebellum, brain stem, hippocampus, medial septum, frontal neocortex, and olfactory bulb. Moreover, we reported the first complete study of Pgk1 expression throughout the development and the aging of mouse brain. Although no tested gene showed to be the optimal reference for all mouse brain regions, in general, the new housekeeping genes were highly stable in most of the analyzed regions. Above all, with few exceptions, Pgk1 showed to be a reliable control for the analyzed mouse brain regions during development, maturation, and aging.

The GPR17 receptor in NG2 expressing cells: Focus on in vivocell maturation and participation in acute trauma and chronic damage

NG2‐expressing cells comprise a population of cycling precursors that can exit the cell cycle and differentiate into mature oligodendrocytes. As a whole, they display heterogeneous properties and behaviors that remain unresolved at the molecular level, although partly interpretable as distinct maturation stages. To address this issue, we analyzed the expression of the GPR17 receptor, recently shown to decorate NG2‐expressing cells and to operate as an early sensor of brain damage, in immature and adult oligodendrocyte progenitors in the intact brain and after injury. In both the early postnatal and adult cerebral cortex, distinct GPR17 protein localizations and expression levels define different stages of oligodendroglial maturation, ranging from the precursor phase to the premyelinating phenotype. As soon as cells exit mitosis, a fraction of NG2‐expressing cells displays accumulation of GPR17 protein in the Golgi apparatus. GPR17 expression is subsequently upregulated and distributed to processes of cells that stop dividing, progressively lose NG2 positivity and assume premyelinating features. Absence of colabeling with mature markers or myelin proteins indicates that GPR17 is downregulated when cells complete their final maturation. BrdU‐based fate‐mapping demonstrated that a significant fraction of newly generated oligodendrocyte progenitors transiently upregulates GPR17 during maturation. Importantly, we also found that GPR17 does not participate to the early reaction of NG2‐expressing cells to damage, while it is induced at postacute stages after injury. These findings identify GPR17 as a marker for progenitor progression within the oligodendroglial lineage and highlight its participation to postacute reactivity of NG2 cells in different injury paradigms.

Distinct Roles of Nogo-A and Nogo Receptor 1 in the Homeostatic Regulation of Adult Neural Stem Cell Function and Neuroblast Migration

In the adult mammalian subventricular zone (SVZ), GFAP-positive neural stem cells (NSCs) generate neuroblasts that migrate tangentially along the rostral migratory stream (RMS) toward the olfactory bulb (OB). In the mouse brain, we found that the plasticity inhibitors Nogo-A and Nogo receptor 1 (NgR1) are differentially expressed in the SVZ–OB system, in which Nogo-A identifies immature neuroblasts and NgR1 germinal astrocytes. We therefore examined the role of Nogo-A and NgR1 in the regulation of neurogenesis. Pharmacological experiments show that Nogo-66/NgR1 interaction reduces the proliferation of NSCs. This is consistent with a negative-feedback loop, in which newly generated neurons modulate cell division of SVZ stem cells. Moreover, the Nogo-A–Δ20 domain promotes neuroblast migration toward the OB through activation of the Rho/ROCK (Rho-associated, coiled-coil containing protein kinase) pathway, without the participation of NgR1. Our findings reveal a new unprecedented function for Nogo-A and NgR1 in the homeostatic regulation of the pace of neurogenesis in the adult mouse SVZ and in the migration of neuroblasts along the RMS.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death.

The P2Y‐like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time‐dependent increase in the number of highly branched GPR17‐positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4‐positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4‐positive cells. Under both culture protocols, appearance of more mature CNPase‐ and MBP‐positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide‐induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double‐edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.

Excitability and synaptic alterations in the cerebellum of APP/PS1 mice.

In Alzheimers disease (AD), the severity of cognitive symptoms is better correlated with the levels of soluble amyloid-beta (Aβ) rather than with the deposition of fibrillar Aβ in amyloid plaques. In APP/PS1 mice, a murine model of AD, at 8 months of age the cerebellum is devoid of fibrillar Aβ, but dosage of soluble Aβ1–42, the form which is more prone to aggregation, showed higher levels in this structure than in the forebrain. Aim of this study was to investigate the alterations of intrinsic membrane properties and of synaptic inputs in Purkinje cells (PCs) of the cerebellum, where only soluble Aβ is present. PCs were recorded by whole-cell patch-clamp in cerebellar slices from wild-type and APP/PS1 mice. In APP/PS1 PCs, evoked action potential discharge showed enhanced frequency adaptation and larger afterhyperpolarizations, indicating a reduction of the intrinsic membrane excitability. In the miniature GABAergic postsynaptic currents, the largest events were absent in APP/PS1 mice and the interspike intervals distribution was shifted to the left, but the mean amplitude and frequency were normal. The ryanodine-sensitive multivescicular release was not altered and the postsynaptic responsiveness to a GABAA agonist was intact. Climbing fiber postsynaptic currents were normal but their short-term plasticity was reduced in a time window of 100–800 ms. Parallel fiber postsynaptic currents and their short-term plasticity were normal. These results indicate that, in the cerebellar cortex, chronically elevated levels of soluble Aβ1–42 are associated with alterations of the intrinsic excitability of PCs and with alterations of the release of GABA from interneurons and of glutamate from climbing fibers, while the release of glutamate from parallel fibers and all postsynaptic mechanisms are preserved. Thus, soluble Aβ1–42 causes, in PCs, multiple functional alterations, including an impairment of intrinsic membrane properties and synapse-specific deficits, with differential consequences even in different subtypes of glutamatergic synapses.

Early phenotypic asymmetry of sister oligodendrocyte progenitor cells after mitosis and its modulation by aging and extrinsic factors

Oligodendrocyte progenitor cells (OPCs) persist in the adult central nervous system and guarantee oligodendrocyte turnover throughout life. It remains obscure how OPCs avoid exhaustion during adulthood. Similar to stem cells, OPCs could self‐maintain by undergoing asymmetric divisions generating a mixed progeny either keeping a progenitor phenotype or proceeding to differentiation. To address this issue, we examined the distribution of stage‐specific markers in sister OPCs during mitosis and later after cell birth, and assessed its correlation with distinct short‐term fates. In both the adult and juvenile cerebral cortex a fraction of dividing OPCs gives rise to sister cells with diverse immunophenotypic profiles and short‐term behaviors. Such heterogeneity appears as cells exit cytokinesis, but does not derive from the asymmetric segregation of molecules such as NG2 or PDGFRa expressed in the mother cell. Rather, rapid downregulation of OPC markers and upregulation of molecules associated with lineage progression contributes to generate early sister OPC asymmetry. Analyses during aging and upon exposure to physiological (i.e., increased motor activity) and pathological (i.e., trauma or demyelination) stimuli showed that both intrinsic and environmental factors contribute to determine the fraction of symmetric and asymmetric OPC pairs and the phenotype of the OPC progeny as soon as cells exit mitosis. GLIA 2015;63:271–286

Beyond cell replacement: unresolved roles of NG2-expressing progenitors

NG2-expressing parenchymal precursors (NG2+p) serve as primary source of myelinating oligodendrocytes in both the developing and adult Central Nervous System (CNS). However, their abundance, limited differentiation potential at adult stages along with stereotypic reaction to injury independent of the extent of myelin loss suggest that NG2+p exert functions additional to myelin production. In support of this view, NG2+p express a complex battery of molecules known to exert neuromodulatory and neuroprotective functions. Further, they establish intimate physical associations with the other CNS cell types, receive functional synaptic contacts and possess ion channels apt to constantly sense the electrical activity of surrounding neurons. These latter features could endow NG2+p with the capability to affect neuronal functions with potential homeostatic outcomes. Here we summarize and discuss current evidence favoring the view that NG2+p can participate in circuit formation, modulate neuronal activity and survival in the healthy and injured CNS, and propose perspectives for studies that may complete our understanding of NG2+p roles in physiology and pathology.

Early Enriched Environment Exposure Protects Spatial Memory and Accelerates Amyloid Plaque Formation in APPSwe/PS1L166P Mice

Enriched environment exposure improves several aspects of cognitive performance in Alzheimer’s disease patients and in animal models and, although the role of amyloid plaques is questionable, several studies also assessed their response to enriched environment, with contrasting results. Here we report that rearing APPSwe/PS1L166P mice in an enriched environment since birth rescued the spatial memory impairment otherwise present at 6 months of age. At the same time, the exposure to the enriched environment caused a transient acceleration of plaque formation, while there was no effect on intracellular staining with the 6E10 antibody, which recognizes β-amyloid, full length amyloid precursor protein and its C-terminal fragments. The anticipation of plaque formation required exposure during early development, suggesting an action within critical periods for circuits formation. On the other hand, chronic neuronal activity suppression by tetrodotoxin decreased the number of plaques without affecting intracellular amyloid. These results indicate that enriched environment exposure since early life has a protective effect on cognitive deterioration although transiently accelerates amyloid deposition. In addition, the effects of the enriched environment might be due to increased neuronal activity, because plaques were reduced by suppression of electrical signaling by tetrodotoxin.

Mouse brain expression patterns of Spg7, Afg3l1, and Afg3l2 transcripts, encoding for the mitochondrial m-AAA protease

BackgroundThe m-AAA (A TPases A ssociated with a variety of cellular A ctivities) is an evolutionary conserved metalloprotease complex located in the internal mitochondrial membrane. In the mouse, it is a hetero-oligomer variably formed by the Spg7, Afg3l1, and Afg3l2 encoded proteins, or a homo-oligomer formed by either Afg3l1 or Afg3l2. In humans, AFG3L2 and SPG7 genes are conserved, whereas AFG3L1 became a pseudogene. Both AFG3L2 and SPG7 are involved in a neurodegenerative disease, namely the autosomal dominant spinocerebellar ataxia SCA28 and a recessive form of spastic paraplegia, respectively.ResultsUsing quantitative RT-PCR, we measured the expression levels of Spg7, Afg3l1, and Afg3l2 in the mouse brain. In all regions Afg3l2 is the most abundant transcript, followed by Spg7, and Afg3l1, with a ratio of approximately 5:3:1 in whole-brain mRNA. Using in-situ hybridization, we showed that Spg7, Afg3l1 and Afg3l2 have a similar cellular pattern of expression, with high levels in mitral cells, Purkinje cells, deep cerebellar nuclei cells, neocortical and hippocampal pyramidal neurons, and brainstem motor neurons. However, in some neuronal types, differences in the level of expression of these genes were present, suggesting distinct degrees of contribution of their proteins.ConclusionsNeurons involved in SCA28 and hereditary spastic paraplegia display high levels of expression, but similar or even higher expression is also present in other types of neurons, not involved in these diseases, suggesting that the selective cell sensitivity should be attributed to other, still unknown, mechanisms.