Loss of function of the mitochondrial peptidase PITRM1 induces proteotoxic stress and Alzheimer's disease-like pathology in human cerebral organoids.

Mutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressing syndrome characterized by cerebellar ataxia, psychotic episodes, and obsessive behavior, as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1-knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPRmt) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid ß in PITRM1-knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the other hand, over time, cerebral organoids generated from PITRM1-knockout iPSCs spontaneously developed pathological features of Alzheimer's disease (AD), including the accumulation of protein aggregates, tau pathology, and neuronal cell death. Single-cell RNA sequencing revealed a perturbation of mitochondrial function in all cell types in PITRM1-knockout cerebral organoids, whereas immune transcriptional signatures were substantially dysregulated in astrocytes. Importantly, we provide evidence of a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. Here, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPRmt that, in turn, modulates cytosolic quality control pathways. Thus, our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.

Alpha-synuclein pathology in the "weaver" mouse, a genetic model of dopaminergic denervation.

Alpha-synuclein plays a pivotal role in Parkinson's disease (PD) pathogenesis, with α-synuclein aggregates/oligomers being identified as toxic species and phosphorylation at Serine 129 promoting aggregation/oligomerization. We investigated the biochemical profile of α-synuclein in the "weaver" mouse, a genetic PD model. Our results revealed increased Serine 129 phosphorylation in the midbrain, striatum, and cortex at a phase of established dopaminergic degeneration on postnatal day 100. These results indicate α-synuclein pathology already at this stage and the potential for age-related progress. Our findings confirm that the "weaver" mouse is an invaluable genetic model to study α-synuclein pathogenesis during PD progression.

Microglial expression of CD83 governs cellular activation and restrains neuroinflammation in experimental autoimmune encephalomyelitis.

Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.

Loss of Complement Factor H impairs antioxidant capacity and energy metabolism of human RPE cells.

Polymorphisms in the Complement Factor H (CFH) gene, coding for the Factor H protein (FH), can increase the risk for age-related macular degeneration (AMD). AMD-associated CFH risk variants, Y402H in particular, impair FH function leading to complement overactivation. Whether this alone suffices to trigger AMD pathogenesis remains unclear. In AMD, retinal homeostasis is compromised due to the dysfunction of retinal pigment epithelium (RPE) cells. To investigate the impact of endogenous FH loss on RPE cell balance, we silenced CFH in human hTERT-RPE1 cells. FH reduction led to accumulation of C3, at both RNA and protein level and increased RPE vulnerability toward oxidative stress. Mild hydrogen-peroxide exposure in combination with CFH knock-down led to a reduction of glycolysis and mitochondrial respiration, paralleled by an increase in lipid peroxidation, which is a key aspect of AMD pathogenesis. In parallel, cell viability was decreased. The perturbations of energy metabolism were accompanied by transcriptional deregulation of several glucose metabolism genes as well as genes modulating mitochondrial stability. Our data suggest that endogenously produced FH contributes to transcriptional and metabolic homeostasis and protects RPE cells from oxidative stress, highlighting a novel role of FH in AMD pathogenesis.

Anti-neuroinflammatory, protective effects of the synthetic microneurotrophin BNN-20 in the advanced dopaminergic neurodegeneration of "weaver" mice.

BNN-20 is a synthetic microneurotrophin, long-term (P1-P21) administration of which exerts potent neuroprotective effect on the "weaver" mouse, a genetic model of progressive, nigrostriatal dopaminergic degeneration. The present study complements and expands our previous work, providing evidence that BNN-20 fully protects the dopaminergic neurons even when administration begins at a late stage of dopaminergic degeneration (>40%). Since neuroinflammation plays a critical role in Parkinson's disease, we investigated the possible anti-neuroinflammatory mechanisms underlying the pharmacological action of BNN-20. The latter was shown to be microglia-mediated, at least in part. Indeed, BNN-20 induced a partial, but significant, reversal of microglia hyperactivation, observed in the untreated "weaver" mouse. Furthermore, it induced a shift in microglia polarization towards the neuroprotective M2 phenotype, suggesting a possible beneficial shifting of microglia activity. This observation was further supported by morphometric measurements. Moreover, BDNF levels, which were severely reduced in the "weaver" mouse midbrain, were restored to normal even after short-term BNN-20 administration. Experiments in "weaver"/NGL (dual GFP/luciferase-NF-κВ reporter) mice using bioluminescence after a short BNN-20 treatment (P60-P74), have shown that the increase of BDNF production was specifically mediated through the TrkB-PI3K-Akt-NF-κB signaling pathway. Interestingly, long-term BNN-20 treatment (P14-P60) significantly increased dopamine levels in the "weaver" striatum, which seems to be associated with the improved motor activity observed in the treated mutant animals. In conclusion, our findings suggest that BNN-20 may serve as a lead molecule for new therapeutic compounds for Parkinson's disease, combining strong anti-neuroinflammatory and neuroprotective properties, leading to elevated dopamine levels and improved motor activity.

Interferon-γ signaling synergizes with LRRK2 in neurons and microglia derived from human induced pluripotent stem cells.

Parkinson's disease-associated kinase LRRK2 has been linked to IFN type II (IFN-γ) response in infections and to dopaminergic neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ remains unclear. In this study, we employed dopaminergic neurons and microglia differentiated from patient-derived induced pluripotent stem cells carrying LRRK2 G2019S, the most common Parkinson's disease-associated mutation. We show that IFN-γ enhances the LRRK2 G2019S-dependent negative regulation of AKT phosphorylation and NFAT activation, thereby increasing neuronal vulnerability to immune challenge. Mechanistically, LRRK2 G2019S suppresses NFAT translocation via calcium signaling and possibly through microtubule reorganization. In microglia, LRRK2 modulates cytokine production and the glycolytic switch in response to IFN-γ in an NFAT-independent manner. Activated LRRK2 G2019S microglia cause neurite shortening, indicating that LRRK2-driven immunological changes can be neurotoxic. We propose that synergistic LRRK2/IFN-γ activation serves as a potential link between inflammation and neurodegeneration in Parkinson's disease.

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC and Fly Models of Parkinson's Disease.

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.

BNN-20, a synthetic microneurotrophin, strongly protects dopaminergic neurons in the "weaver" mouse, a genetic model of dopamine-denervation, acting through the TrkB neurotrophin receptor.

Neurotrophic factors are among the most promising treatments aiming at slowing or stopping and even reversing Parkinson's disease (PD). However, in most cases, they cannot readily cross the human blood-brain-barrier (BBB). Herein, we propose as a therapeutic for PD the small molecule 17-beta-spiro-[5-androsten-17,2'-oxiran]-3beta-ol (BNN-20), a synthetic analogue of DHEA, which crosses the BBB and is deprived of endocrine side-effects. Using the "weaver" mouse, a genetic model of PD, which exhibits progressive dopaminergic neurodegeneration in the Substantia Nigra (SN), we have shown that long-term administration (P1-P21) of BNN-20 almost fully protected the dopaminergic neurons and their terminals, via i) a strong anti-apoptotic effect, probably mediated through the Tropomyosin receptor kinase B (TrkB) neurotrophin receptor's PI3K-Akt-NF-κB signaling pathway, ii) by exerting an efficient antioxidant effect, iii) by inducing significant anti-inflammatory activity and iv) by restoring Brain-Derived Neurotrophic Factor (BDNF) levels. By intercrossing "weaver" with NGL mice (dual GFP/luciferase-NF-κΒ reporter mice, NF-κΒ.GFP.Luc), we obtained Weaver/NGL mice that express the NF-κB reporter in all somatic cells. Acute BNN-20 administration to Weaver/NGL mice induced a strong NF-κB-dependent transcriptional response in the brain as detected by bioluminescence imaging, which was abolished by co-administration of the TrkB inhibitor ANA-12. This indicates that BNN-20 exerts its beneficial action (at least in part) through the TrkB-PI3K-Akt-NF-κB signaling pathway. These results could be of clinical relevance, as they suggest BNN-20 as an important neuroprotective agent acting through the TrkB neurotrophin receptor pathway, mimicking the action of the endogenous neurotrophin BDNF. Thus BNN-20 could be proposed for treatment of PD.