ER-mitochondria contact sites in neurodegeneration: genetic screening approaches to investigate novel disease mechanisms.

Funder: Open Targets OTAR050 UK Dementia Research Institute RRZA/175Mitochondria-ER contact sites (MERCS) are known to underpin many important cellular homoeostatic functions, including mitochondrial quality control, lipid metabolism, calcium homoeostasis, the unfolded protein response and ER stress. These functions are known to be dysregulated in neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD) and amyloid lateral sclerosis (ALS), and the number of disease-related proteins and genes being associated with MERCS is increasing. However, many details regarding MERCS and their role in neurodegenerative diseases remain unknown. In this review, we aim to summarise the current knowledge regarding the structure and function of MERCS, and to update the field on current research in PD, AD and ALS. Furthermore, we will evaluate high-throughput screening techniques, including RNAi vs CRISPR/Cas9, pooled vs arrayed formats and how these could be combined with current techniques to visualise MERCS. We will consider the advantages and disadvantages of each technique and how it can be utilised to uncover novel protein pathways involved in MERCS dysfunction in neurodegenerative diseases

Amyloid Precursor Protein Is Trafficked and Secreted via Synaptic Vesicles

A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling

Mitochondrial division inhibitor-1 is neuroprotective in the A53T-α-synuclein rat model of Parkinson’s disease

Alpha-synuclein (α-syn) is involved in both familial and sporadic Parkinson’s disease (PD). One of the proposed pathogenic mechanisms of α-syn mutations is mitochondrial dysfunction. However, it is not entirely clear the impact of impaired mitochondrial dynamics induced by α-syn on neurodegeneration and whether targeting this pathway has therapeutic potential. In this study we evaluated whether inhibition of mitochondrial fission is neuroprotective against α-syn overexpression in vivo. To accomplish this goal, we overexpressed human A53T-α- synuclein (hA53T-α-syn) in the rat nigrostriatal pathway, with or without treatment using the small molecule Mitochondrial Division Inhibitor-1 (mdivi-1), a putative inhibitor of the mitochondrial fission Dynamin-Related Protein-1 (Drp1). We show here that mdivi-1 reduced neurodegeneration, α-syn aggregates and normalized motor function. Mechanistically, mdivi-1 reduced mitochondrial fragmentation, mitochondrial dysfunction and oxidative stress. These in vivo results support the negative role of mutant α-syn in mitochondrial function and indicate that mdivi-1 has a high therapeutic potential for PD

A Bi-fluorescence complementation system to detect associations between the Endoplasmic reticulum and mitochondria

Abstract Close contacts between the endoplasmic reticulum membrane and the mitochondrial outer membrane facilitate efficient transfer of lipids between the organelles and coordinate Ca2+ signalling and stress responses. Changes to this coupling is associated with a number of metabolic disorders and neurodegenerative diseases including Alzheimer’s, Parkinson’s and motor neuron disease. The distance between the two membranes at regions of close apposition is below the resolution of conventional light microscopy, which makes analysis of these interactions challenging. Here we describe a new bifluorescence complementation (BiFC) method that labels a subset of ER-mitochondrial associations in fixed and living cells. The total number of ER-mitochondria associations detected by this approach increases in response to tunicamycin-induced ER stress, serum deprivation or reduced levels of mitofusin 2 (MFN2). This method will facilitate the analysis of dynamic interactions between the ER and mitochondrial membranes

PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial–endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome

Lipid level alteration in human and cellular models of alpha synuclein mutations

Lipid profiles in biological fluids from patients with Parkinson’s disease (PD) are increasingly investigated in search of biomarkers. However, the lipid profiles in genetic PD remain to be determined, a gap of knowledge of particular interest in PD associated with mutant α-synuclein (SNCA), given the known relationship between this protein and lipids. The objective of this research is to identify serum lipid composition from SNCA A53T mutation carriers and to compare these alterations to those found in cells and transgenic mice carrying the same genetic mutation. We conducted an unbiased lipidomic analysis of 530 lipid species from 34 lipid classes in serum of 30 participants with SNCA mutation with and without PD and 30 healthy controls. The primary analysis was done between 22 PD patients with SNCA+ (SNCA+/PD+) and 30 controls using machine-learning algorithms and traditional statistics. We also analyzed the lipid composition of human clonal-cell lines and tissue from transgenic mice overexpressing the same SNCA mutation. We identified specific lipid classes that best discriminate between SNCA+/PD+ patients and healthy controls and found certain lipid species, mainly from the glycerophosphatidylcholine and triradylglycerol classes, that are most contributory to this discrimination. Most of these alterations were also present in human derived cells and transgenic mice carrying the same mutation. Our combination of lipidomic and machine learning analyses revealed alterations in glycerophosphatidylcholine and triradylglycerol in sera from PD patients as well as cells and tissues expressing mutant α-Syn. Further investigations are needed to establish the pathogenic significance of these α-Syn-associated lipid changes. © 2022, The Author(s)