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-progressive 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 β in PITRM1 knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the contrary, cerebral organoids generated from PITRM1 knockout iPSCs spontaneously developed over time pathological features of Alzheimer’s disease (AD), including accumulation of protein aggregates, tau pathology, and neuronal cell death. Importantly, we provide evidence for a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. In summary, 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.

Viia7 Real time PCR system (Applied Biosystems). The expression level of each 2 4 0 gene was normalized to the housekeeping gene ribosomal protein large P0 (Rplp0).

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Fold-changes in gene expression were calculated using the 2 -DDCT method, based on 2 4 2 biological reference samples and housekeeping genes for normalization. treatment with lysosomal inhibitor for 4h, by the levels of LC3-II without treatment. Caspase 3 (1:500, Cell Signaling Technology 9664T). LC3 particle number in β -III-2 9 3 tubulin positive cells was quantified with the "analyse particles" plug-in in ImageJ 2 9 4 (NIH). Caspase 3 was quantified with mean fluorescence intensity in Image J (NIH).

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Quantification was carried out on, at least, 50 cells per condition, from three 2 9 6 independent experiments. The LDH assay (Promega) was performed as per the manufacturer's instructions. For amyloid-beta species measurement in iPSC-cortical neurons, 10^6 cells were 3 0 3 plated into a one well of 12-well plate and cell supernatant was collected after 5 days, Individual cerebral organoids were homogenized in ice-cold RIPA buffer containing and 4°C for 10min. The protein concentration of the supernatant was determined by using ELISA assays (both Invitrogen, KHB0041 and KHO0631, respectively) according to manufacturer's instructions. was used to analyze the data. Statistical significance was evaluated using two-tailed  In order to overcome the limitation of the embryonic lethality previously observed in homozygous clones were generated, and two fully characterized clones were DNA ratio (Fig. 2I). Taken together, these results suggest that loss of PITRM1 To examine the impact of PITRM1 activity on Aβ pathology, we first assessed the were not detected in mitochondrial extracts from PITRM1 deficient neurons. Next, we turnover 23 , we examined the levels of ubiquitinated proteins by Western blot. We  neuronal cultures did not show Aβ aggregates nor tau pathology or overt cell death.
Interestingly, ISRIB-treated cerebral organoids also showed an increase of mitochondrial clearance (Fig. 5E). Enhancing mitophagy via NAD+ precursors ameliorates Aβ proteotoxicity 4 7 5 Since defects in mitophagy have been shown to contribute to AD 29 and ISRIB-  organoids after treatment (Fig. 5F). These data suggest a differential role of NAD+ studied. To address these quetions, we have generated a novel human stem cell- diseases. Employing iPSC-derived cortical neurons, we found that loss of PITRM1 methods coupled with a highly sensitive immunoassay, we were unable to detect Aβ autophagy/mitophagy) that ensure the maintenance of cellular proteostasis. However, over time, these mechanisms may be not sufficient to protect neuronal cells against 5 7 4 mitochondrial proteotoxicity, as observed in long-term culture cerebral organoids. Several findings, including the induction of autophagic flux, decreased mtDNA levels, and increase of mitochondrial protein ubiquitination suggest that PITRM1 deficiency 5 7 8 leads to increased mitochondrial clearance. It is known that defects in PITRM1 mitochondrial clearance in our model. Interestingly, mitochondrial stress response and mitophagy transcripts have been mitophagy is able to rescue AD-related pathology in different AD model systems 29 .

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In line with this finding, we show that stimulating mitophagy with NMN, a NAD+ 5 9 5 booster, significantly improves mitochondrial clearance, with a reduction of 5 9 6 Aβ42/Aβ40 ratio and tau hyperphosphorylation. On the contrary, inhibition of UPR mt 5 9 7 with ISRIB led to decreased mitochondrial clearance and aggravation of Aβ and tau  In conclusion, we report a novel cellular model of human PITRM1 deficiency that hyperphosphorylation, and neuronal death. We report that PITRM1 deficiency 6 1 0 induces impairment of mitochondrial proteostasis and activation of UPR mt that 6 1 1 activates cytosolic quality control pathways, such as the UPS and autophagy. The rare, the disease mechanisms described in the present study may apply to both 6 1 9 primary mitochondrial diseases and more common adult-onset neurological diseases.