Iron overload causes endolysosomal deficits modulated by NAADP-regulated two pore channels and RAB7A

Various neurodegenerative disorders are associated with increased brain iron content. Iron is known to cause oxidative stress, which concomitantly promotes cell death. Whereas endolysosomes are known to serve as intracellular iron storage organelles, the consequences of increased iron on endolysosomal functioning, and effects on cell viability upon modulation of endolysosomal iron release remain largely unknown. Here, we show that increasing intracellular iron causes endolysosomal alterations associated with impaired autophagic clearance of intracellular protein aggregates, increased cytosolic oxidative stress and increased cell death. These effects are subject to regulation by NAADP, a potent second messenger reported to target endolysosomal TPCNs (2-pore channels). Consistent with endolysosomal iron storage, cytosolic iron levels are modulated by NAADP, and increased cytosolic iron is detected when overexpressing active, but not inactive TPCNs, indicating that these channels can modulate endolysosomal iron release. Cell death triggered by altered intralysosomal iron handling is abrogated in the presence of an NAADP antagonist or when inhibiting RAB7A activity. Taken together, our results suggest that increased endolysosomal iron causes cell death associated with increased cytosolic oxidative stress as well as autophagic impairments, and these effects are subject to modulation by endolysosomal ion channel activity in a RAB7A-dependent manner. These data highlight alternative therapeutic strategies for neurodegenerative disorders associated with increased intracellular iron load

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by “tethering proteins” that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson’s disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson’s disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson’s disease and FTD/ALS

Analysis of LRRK2 Towards understanding the pathogenic mechanisms underlying parkinson's disease: Deregulated autophagy and endocytosis

Although the majority of PD cases are idiopathic, the identification of diseasecausing mutations helps in our understanding of the molecular mechanisms involved in neuronal demise. Mutations in LRRK2 (leucine-rich repeat kinase 2) are found associated with both sporadic and familial Parkinson´s disease (PD). LRRK2 is a multidomain protein characterised by kinase and GTPase activities, with pathogenic mutations localized to both catalytic domains. There is a significant body of evidence correlating altered catalytic activity with cytotoxicity. However, early cellular LRRK2- mediated events which eventually lead to cellular demise remain poorly understood. LRRK2 has been implicated in autophagic and endosomal trafficking pathways in vitro and in vivo, even though the mechanism(s) remain unclear. This thesis describes work towards addressing how pathogenic LRRK2 may cause cellular dysfunction linked to PD pathogenesis, with emphasis on autophagy and endosomal trafficking deficits. First, mutant LRRK2 was found to cause a block in autophagic flux through a pathway involving NAADP (nicotinic acid adenine dinucleotide phosphate)-sensitive channels in acidic late endosomal/lysosomal structures. Pathogenic LRRK2 was found to cause a partial increase in lysosomal pH, and decreased cell survival in the presence of protein aggregation-induced stress. Since autophagy and endocytosis share late endosomes/lysosomes as common end-points, the effect of mutant LRRK2 on endocytosis was evaluated. Pathogenic LRRK2 was found to cause deficits in both early and late events of receptor-mediated endocytosis, including a delay in receptor trafficking out of late endosomes, which become aberrantly elongated. Rab7 is a small GTPase involved in various endosomal trafficking processes, including late endosomal-lysosomal trafficking and retromer-mediated trafficking between late endosomes and the trans-Golgi network. The effects on receptor trafficking could be rescued when overexpressing constitutively active Rab7 or Rab7L1, as well as the Rab7-interacting proteins dynamin2/CIN85, which localize to late endosomes and regulate degradative receptor trafficking. Pathogenic LRRK2 expression was associated with a decrease in Rab7 activity. Alltogether, the present results highlight an important role for pathogenic LRRK2 in deregulating several endolysosomal membrane trafficking events, which may underlie early cellular pathogenesis in PD.Tesis Univ. Granada. Programa Oficial de Doctorado en: Biomedicina Regenerativ

Analysis of LRRK2 Towards understanding the pathogenic mechanisms underlying parkinson's disease: Deregulated autophagy and endocytosis

Although the majority of PD cases are idiopathic, the identification of diseasecausing mutations helps in our understanding of the molecular mechanisms involved in neuronal demise. Mutations in LRRK2 (leucine-rich repeat kinase 2) are found associated with both sporadic and familial Parkinson´s disease (PD). LRRK2 is a multidomain protein characterised by kinase and GTPase activities, with pathogenic mutations localized to both catalytic domains. There is a significant body of evidence correlating altered catalytic activity with cytotoxicity. However, early cellular LRRK2- mediated events which eventually lead to cellular demise remain poorly understood. LRRK2 has been implicated in autophagic and endosomal trafficking pathways in vitro and in vivo, even though the mechanism(s) remain unclear. This thesis describes work towards addressing how pathogenic LRRK2 may cause cellular dysfunction linked to PD pathogenesis, with emphasis on autophagy and endosomal trafficking deficits. First, mutant LRRK2 was found to cause a block in autophagic flux through a pathway involving NAADP (nicotinic acid adenine dinucleotide phosphate)-sensitive channels in acidic late endosomal/lysosomal structures. Pathogenic LRRK2 was found to cause a partial increase in lysosomal pH, and decreased cell survival in the presence of protein aggregation-induced stress. Since autophagy and endocytosis share late endosomes/lysosomes as common end-points, the effect of mutant LRRK2 on endocytosis was evaluated. Pathogenic LRRK2 was found to cause deficits in both early and late events of receptor-mediated endocytosis, including a delay in receptor trafficking out of late endosomes, which become aberrantly elongated. Rab7 is a small GTPase involved in various endosomal trafficking processes, including late endosomal-lysosomal trafficking and retromer-mediated trafficking between late endosomes and the trans-Golgi network. The effects on receptor trafficking could be rescued when overexpressing constitutively active Rab7 or Rab7L1, as well as the Rab7-interacting proteins dynamin2/CIN85, which localize to late endosomes and regulate degradative receptor trafficking. Pathogenic LRRK2 expression was associated with a decrease in Rab7 activity. Alltogether, the present results highlight an important role for pathogenic LRRK2 in deregulating several endolysosomal membrane trafficking events, which may underlie early cellular pathogenesis in PD.Tesis Univ. Granada. Programa Oficial de Doctorado en: Biomedicina Regenerativ

S: A link between LRRK2, autophagy and NAADP-mediated endolysosomal calcium signalling. Biochem Soc Trans 2012

Abstract Mutations in LRRK2 (leucine-rich repeat kinase 2) represent a significant component of both sporadic and familial PD (Parkinson's disease). Pathogenic mutations cluster in the enzymatic domains of LRRK2, and kinase activity seems to correlate with cytotoxicity, suggesting the possibility of kinase-based therapeutic strategies for LRRK2-associated PD. Apart from cytotoxicity, changes in autophagy have consistently been observed upon overexpression of mutant, or knockdown of endogenous, LRRK2. However, delineating the precise mechanism(s) by which LRRK2 regulates autophagy has been difficult. Recent data suggest a mechanism involving late steps in autophagic-lysosomal clearance in a manner dependent on NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive lysosomal Ca 2 + channels. In the present paper, we review our current knowledge of the link between LRRK2 and autophagic-lysosomal clearance, including regulation of Ca 2 + -dependent events involving NAADP

ER–mitochondria signaling in Parkinson’s disease

International audienceMitochondria form close physical contacts with a specialized domain of the endoplasmic reticulum (ER), known as the mitochondria-associated membrane (MAM). This association constitutes a key signaling hub to regulate several fundamental cellular processes. Alterations in ER–mitochondria signaling have pleiotropic effects on a variety of intracellular events resulting in mitochondrial damage, Ca 2+ dyshomeostasis, ER stress and defects in lipid metabolism and autophagy. Intriguingly, many of these cellular processes are perturbed in neurodegenerative diseases. Furthermore, increasing evidence highlights that ER–mitochondria signaling contributes to these diseases, including Parkinson's disease (PD). PD is the second most common neurodegenerative disorder, for which effective mechanism-based treatments remain elusive. Several PD-related proteins localize at mitochondria or MAM and have been shown to participate in ER–mitochondria signaling regulation. Likewise, PD-related mutations have been shown to damage this signaling. Could ER–mitochondria associations be the link between pathogenic mechanisms involved in PD, providing a common mechanism? Would this provide a pharmacological target for treating this devastating disease? In this review, we aim to summarize the current knowledge of ER–mitochondria signaling and the recent evidence concerning damage to this signaling in PD

Mitochondrial Dysfunction in Repeat Expansion Diseases

Repeat expansion diseases are a group of neuromuscular and neurodegenerative disorders characterized by expansions of several successive repeated DNA sequences. Currently, more than 50 repeat expansion diseases have been described. These disorders involve diverse pathogenic mechanisms, including loss-of-function mechanisms, toxicity associated with repeat RNA, or repeat-associated non-ATG (RAN) products, resulting in impairments of cellular processes and damaged organelles. Mitochondria, double membrane organelles, play a crucial role in cell energy production, metabolic processes, calcium regulation, redox balance, and apoptosis regulation. Its dysfunction has been implicated in the pathogenesis of repeat expansion diseases. In this review, we provide an overview of the signaling pathways or proteins involved in mitochondrial functioning described in these disorders. The focus of this review will be on the analysis of published data related to three representative repeat expansion diseases: Huntington’s disease, C9orf72-frontotemporal dementia/amyotrophic lateral sclerosis, and myotonic dystrophy type 1. We will discuss the common effects observed in all three repeat expansion disorders and their differences. Additionally, we will address the current gaps in knowledge and propose possible new lines of research. Importantly, this group of disorders exhibit alterations in mitochondrial dynamics and biogenesis, with specific proteins involved in these processes having been identified. Understanding the underlying mechanisms of mitochondrial alterations in these disorders can potentially lead to the development of neuroprotective strategies