Conference Agenda

Parkinson's disease (PD) is characterized by the progressive loss of DAergic neuronal cell bodies in the ventral midbrain (VMB), and their terminals in the striatum (STR). In PD, astrocytes (AS) can have either destructive or beneficial effects. However, the complex intercellular signaling between AS and neurons has not been fully elucidated, yet. Extracellular vesicles (EVs) represent a relevant strategy to transfer information between cells. EVs are membranous nanoparticles containing different molecules such as nucleic acids, proteins, etc. Based on size, EVs are classified as small EVs (e.g., exosomes) and medium/large EVs.

In our recent work, we demonstrated that AS from the VMB and STR release a population of small-EVs (⁓100 nm) in a region-specific manner, with VMB-AS secreting the highest rate of EVs. Functional studies revealed that both VMB- and STR-AS-EVs counteract H₂O₂-induced caspase-3 activation on differentiated SH-SY5Y cells. Also, AS-EVs recover mitochondrial complex I functionality injured by MPP⁺ neurotoxin. Interestingly, only VMB-AS-EVs ameliorate ATP production, supporting a regional specificity in targeting mitochondrial dysfunction.

To investigate the mechanism(s) of neuroprotection exerted by AS-EVs, we are studying: (i) how AS-EVs enter target neurons; and (ii) which are the AS-EV molecular cargoes. Using proteinase k treatment and uptake inhibitors, we observed that surface proteins are crucial for EV entry. On the other hand, the AS-EV content was characterized via small RNA-sequencing and mass spectrometry analysis. Interestingly, the RNA content of VMB-AS-EVs revealed that an important portion (25%) of sRNAs corresponds to tRNA-derived fragments, some of which were recently identified as significantly upregulated in PD patients. MS analysis of VMB-AS-EVs shows the presence of both cytosolic and mitochondrial proteins, related to neuroprotective pathways.

The identification of the molecular players involved in the complex cell-to-cell communication mediated by AS-EVs may pave the way for the development of innovative therapeutic approaches to tackle PD.

Cortical astrocytes show an impressive heterogeneity across mammals. While protoplasmic and fibrous astrocytes have been observed in all mammals, there are two types of astrocytes with special features in primates: the interlaminar astrocytes (ILAs) and the varicose-projection astrocytes (VP-As). The interlaminar astrocytes (ILAs) are a subset of Glial fibrillary acidic protein (GFAP)⁺ astrocytes with singular morphological traits: they can be identified in the cerebral cortex by having a cell body in the most marginal layer of the cerebral cortex (layer I), very close to the pia, and long, interlaminar processes running into deeper cortical layers, reaching layer V in humans. We compared ILA morphology, density, and molecular markers across mammalian evolution and development, and found they have special features in primates. VP-As, instead are a special type of astrocyte observed in hominoid species only. VP-As are usually visible in the deeper layers of the cortex and show peculiar varicosities (beads) along their longest processes. We analyzed the presence and appearance of VP-As across multiple species of primates, with a special focus on apes and humans, we described their distribution and their expression of specific astrocyte markers across species. I will show data resulting from studying these intriguing astrocyte types and will discuss potential relevance for future functional studies of astrocytes in development and evolution.

Mitochondria are considered to be the “powerhouse” of the cells for their ability to generate energy in the form of adenosine triphosphate (ATP) through a process called oxidative phosphorylation (OXPHOS). Glucose-derived pyruvate and fatty acid-derived acetyl-CoA are among the main substrates for OXPHOS. In addition, mitochondria also orchestrate multifaceted cell signalling processes to control cell division, senescence, differentiation, inflammation and cell death¹. In the brain, mitochondria are highly important for neurons, yet considered less so for glial cells such as astrocytes and adult neural stem cells (also called radial glia-like cells). For many years, glial cells were considered glycolytic cells, transforming glucose into lactate rather than using it for OXPHOS. However, recent advances show that these cells have a complex and dynamic mitochondrial network^2,3. In this presentation, I will discuss our recent findings on how modulating the entry of pyruvate or fatty acids into mitochondria strongly influences the activity and the function of both adult radial glial-like cells and astrocytes. Finally, I will present new findings to highlight the importance of mitochondria in the control of astrocyte behavior.

1. Giacomello M et al., 2020, Nature Review Mol. Cell Bio 21, 204-224.

2. Zehnder T., Petrelli F., et al., 2021, Cell Reports 26, 719-737.e6.

3. Petrelli F., et al., 2023, Science advances 9, eadd5220

Under certain conditions, astrocytes demonstrate phagocytic capability and cooperate with microglia as an ancillary clearance system to clear up the brain. Noteworthy, astrocytes are efficient sensors of synaptic dysfunction or degeneration. Indeed, they intervene by eliminating neuronal terminals, engulfing debris and internalizing neuronal-released aggregated proteins. However, the molecular machinery recruited for the recognition of specific targets is only in part clarified. Here, we implemented an in vitro live-imaging protocol to follow synapses engulfment in astrocytes using synaptosomes conjugated with a fluorescent pH indicator. Taking advantage of High Content Imaging system, we dissected the phagocytic process by performing individual gene silencing of a druggable library. We found that atypical chemokine receptor 3 (Ackr3) abolishes synaptosome internalization, thus pointing to chemokines as key signaling factors in synaptic removal. We are currently investigating the direct involvement of Ackr3 in the recognition and internalization of neuronal terminals mediated by murine and human astrocytes using different biochemical and imaging assays. Of note, ACKR3 has been detected in different human pathologies, such as Alzheimer's disease and glioblastoma multiforme. Future studies will be aimed to the study of ACKR3-mediated synaptic elimination in human models as well as in patient biofluids. A successful outcome of this research might help to understand the contribution of astrocyte-mediated clearance to brain disorders.