Ceramide accumulation induces mitophagy and impairs β-oxidation in PINK1 deficiency

Energy production via the mitochondrial electron transport chain (ETC) and mitophagy are two important processes affected in Parkinson’s disease (PD). Interestingly, PINK1, mutations of which cause early-onset PD, plays a key role in both processes, suggesting that these two mechanisms are connected. However, the converging link of both pathways currently remains enigmatic. Recent findings demonstrated that lipid aggregation, along with defective mitochondria, is present in postmortem brains of PD patients. In addition, an increasing body of evidence shows that sphingolipids, including ceramide, are altered in PD, supporting the importance of lipids in the pathophysiology of PD. Here, we identified ceramide to play a crucial role in PINK1-related PD that was previously linked almost exclusively to mitochondrial dysfunction. We found ceramide to accumulate in mitochondria and to negatively affect mitochondrial function, most notably the ETC. Lowering ceramide levels improved mitochondrial phenotypes in pink1-mutant flies and PINK1-deficient patient-derived fibroblasts, showing that the effects of ceramide are evolutionarily conserved. In addition, ceramide accumulation provoked ceramide-induced mitophagy upon PINK1 deficiency. As a result of the ceramide accumulation, β-oxidation in PINK1 mutants was decreased, which was rescued by lowering ceramide levels. Furthermore, stimulation of β-oxidation was sufficient to rescue PINK1-deficient phenotypes. In conclusion, we discovered a cellular mechanism resulting from PD-causing loss of PINK1 and found a protective role of β-oxidation in ETC dysfunction, thus linking lipids and mitochondria in the pathophysiology of PINK1-related PD. Furthermore, our data nominate β-oxidation and ceramide as therapeutic targets for PD.

Loss of PINK1 function causes autosomal recessive early-onset Parkinson’s disease (PD). Most patients present with bradykinesia, rigidity, resting tremor, and dyskinesia and are responsive to dopamine replacement therapy (1). On the cellular level, PINK1 disease mutations result in impaired energy metabolism and a variety of mitochondrial defects that can partially be alleviated by stimulation of energy metabolism (2–4). Intriguingly, abnormal mitochondrial morphology, along with lipid aggregates, was recently discovered to be present in Lewy bodies of postmortem PD patients’ brains (5), challenging the previously held notion of alpha-synuclein being the almost exclusive neuropathological correlate. This finding confirms the involvement of mitochondrial dysfunction in PD and additionally suggests a critical role of lipids in the pathogenesis of PD.PINK1 is important for the phosphorylation of the Complex I subunit NdufA10 resulting in efficient Complex I and electron transport chain (ETC) activity (6, 7). This function is evolutionarily conserved between Drosophila and humans. Hence, in both flies and humans, loss of PINK1 results in an impaired ETC, reduced ATP levels, and defective mitochondrial morphology (6, 8, 9), all of which are ubiquitously observed in the fly already at the early larval stage. Furthermore, alongside Parkin, PINK1 plays a crucial role in mitophagy to remove defective mitochondria that appears to be defective in an age-dependent fashion (10–13). Pink1-mutant Drosophila melanogaster additionally show thorax muscle degeneration and defective flying ability (8, 9). These latter defects, together with impaired mitochondrial morphology, can be rescued by expressing the fission-promoting protein Drp1 (14). However, increased fission does not improve ETC-related defects (15). Furthermore, stimulation or facilitation of the ETC rescues ETC-related phenotypes in pink1-mutant Drosophila, including ATP levels and mitochondrial morphology (3, 4, 7, 15, 16). These data collectively suggest two parallel mechanisms that converge on a shared common pathway leading to the development of PD. However, the link between these two pathways has yet to be resolved.Recently, disrupted lipid homeostasis has garnered increasing attention in PD (16–18). Furthermore, ceramide, the basic sphingolipid, is altered in several PD models and has been implicated in PD-related alpha-synuclein toxicity (17–20). Interestingly, ceramide induces mitophagy that is facilitated by Drp1 (21). Furthermore, pathogenic variants in Glucocerebrosidase (GCase), an enzyme involved in ceramide synthesis, are known to be the most common risk factor for PD (22, 23). However, the exact mechanism remains enigmatic. We found increased ceramide levels in isolated mitochondria of Pink1^−/− knockout (KO) mouse embryonic fibroblasts (MEFs) (16) and Pink1-deficient flies. Increased ceramide levels are detrimental for proper ETC function (24). Hence, we hypothesize that ceramide accumulation in PINK1 deficiency affects ETC function and mitophagy and constitutes the missing link between these two important processes affected in PD.     

Kurarinone alleviated Parkinson's disease via stabilization of epoxyeicosatrienoic acids in animal model

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders and is characterized by loss of dopaminergic neurons in the substantia nigra (SN), causing bradykinesia and rest tremors. Although the molecular mechanism of PD is still not fully understood, neuroinflammation has a key role in the damage of dopaminergic neurons. Herein, we found that kurarinone, a unique natural product from Sophora flavescens, alleviated the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–induced behavioral deficits and dopaminergic neurotoxicity, including the losses of neurotransmitters and tyrosine hydroxylase (TH)–positive cells (SN and striatum [STR]). Furthermore, kurarinone attenuated the MPTP-mediated neuroinflammation via suppressing the activation of microglia involved in the nuclear factor kappa B signaling pathway. The proteomics result of the solvent-induced protein precipitation and thermal proteome profiling suggest that the soluble epoxide hydrolase (sEH) enzyme, which is associated with the neuroinflammation of PD, is a promising target of kurarinone. This is supported by the increase of plasma epoxyeicosatrienoic acids (sEH substrates) and the decrease of dihydroxyeicosatrienoic acids (sEH products), and the results of in vitro inhibition kinetics, surface plasmon resonance, and cocrystallization of kurarinone with sEH revealed that this natural compound is an uncompetitive inhibitor. In addition, sEH knockout (KO) attenuated the progression of PD, and sEH KO plus kurarinone did not further reduce the protection of PD in MPTP-induced PD mice. These findings suggest that kurarinone could be a potential natural candidate for the treatment of PD, possibly through sEH inhibition.

Parkinson’s disease (PD) is the second-most common neurodegenerative disorder after Alzheimer’s disease (AD) and affects 1.7% of the population over 65 y old, especially people over 80 y old (1, 2). PD is caused by the loss of dopaminergic neurons in the substantia nigra (SN), and it is associated with accumulation of Lewy bodies (LBs) in neuronal somata and Lewy neurites in neuronal processes with fibrillar α-synuclein (3). PD is characterized by the classical motor features of parkinsonism, including bradykinesia, rest tremor, and rigidity as well as postural instability (4, 5). Advances have been made in understanding PD neurodegenerative pathophysiology (4–6). However, translation into patients’ care is still lagging well behind. So far, the treatment of PD in recent trials still depends on strategies for neuroprotection, motor symptoms, and nonmotor symptoms (6). However, PD symptoms have proven elusive to slow down or reverse through the aforementioned interventions (6); therefore, a cure for PD (no symptoms, no side effects, to borrow a phrase from the epilepsy field) has not yet been achieved.Although the molecular mechanism resulting in the neuronal degeneration of PD is not fully understood, some factors are associated with the damage of dopaminergic neurons in PD, including mitochondrial dysfunction, oxidative stress, endoplasmic reticulum stress, and especially neuroinflammation (7, 8). Extensive recent evidence revealed that the release of α-synuclein from dopaminergic neurons activated microglia cells to cause the neuroinflammation, allowing the increase of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) (9, 10). These changes have been found in postmortem brain tissue from PD patients (11, 12). Therefore, neuroinflammation plays a central role in the development of PD and is the target of some recent investigations for treating PD (13–16).Natural products are an important resource of innovational drugs since they possess complex and changeable structures and remarkable biological effects. A great body of evidence has indicated the effect of natural products from traditional Chinese medicines in the neuroinflammation of PD (17–19), such as genistein, resveratrol, and alaternin. Kurarinone is one of the major constituents of the traditional Chinese medicine Sophorae Flavescentis Radix, or Kushen in Chinese (the root of Sophora flavescens), which is often used to treat diarrhea, bacterial and fungal infections, eczema, and inflammation-related diseases (20). Kurarinone shares a flavanone core with a characteristic lavandulyl moiety at C-8 (21, 22) and possesses several pharmacological effects, such as anti-inflammatory and antioxidative activities (23, 24), as well as activation on the large-conductance Ca²⁺-activated K⁺ channel (25, 26).Therefore, in this study, we first tested the ability of kurarinone to reduce neuroinflammation and improve behavioral deficits in a PD mice model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To understand how kurarinone decreases inflammation and because its treatment resulted in elevated levels of epoxyeicosatrienoic acids (EETs), endogenous signaling molecules that control inflammation (27), we used several biochemical methods to determine the molecular target of kurarinone. The interactions between kurarinone and soluble epoxide hydrolase, the main enzyme metabolizing EETs (28), were confirmed using enzyme kinetics and cocrystallization. Our findings suggest that kurarinone could be a potential natural candidate for the treatment of PD through sEH inhibition and other mechanisms, as well as being a lead to develop a new family of sEH inhibitors.     

STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson’s disease

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The loss of SNc dopaminergic neurons affects the plasticity of striatal neurons and leads to significant motor and cognitive disabilities during the progression of the disease. PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in genetic and sporadic PD. Mutations in PARK2 are a major contributing factor in the early onset of autosomal-recessive juvenile parkinsonism (AR-JP), although the mechanisms by which a disruption in parkin function contributes to the pathophysiology of PD remain unclear. Here we demonstrate that parkin is an E3 ligase for STEP₆₁ (striatal-enriched protein tyrosine phosphatase), a protein tyrosine phosphatase implicated in several neuropsychiatric disorders. In cellular models, parkin ubiquitinates STEP₆₁ and thereby regulates its level through the proteasome system, whereas clinically relevant parkin mutants fail to do so. STEP₆₁ protein levels are elevated on acute down-regulation of parkin or in PARK2 KO rat striatum. Relevant to PD, STEP₆₁ accumulates in the striatum of human sporadic PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. The increase in STEP₆₁ is associated with a decrease in the phosphorylation of its substrate ERK1/2 and the downstream target of ERK1/2, pCREB [phospho-CREB (cAMP response element-binding protein)]. These results indicate that STEP₆₁ is a novel substrate of parkin, although further studies are necessary to determine whether elevated STEP₆₁ levels directly contribute to the pathophysiology of PD.Parkinson’s disease (PD) is a common motor disorder with clinical symptoms that include bradykinesia, resting tremor, rigidity, postural instability, and cognitive deficits (1–3). The pathophysiology of PD includes selective loss of dopaminergic neurons in the substantia nigra, with a progressive depletion of striatal dopamine and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies. Mutations of several genes are implicated in PD and are responsible for ∼10% of cases; the remaining cases are classified as sporadic PD. Although specific mutations in genes that include PARK2, PINK-1, LRRK2, and DJ-1 are known, the effects these mutations have on intracellular signaling and disease progression are not well understood and form an area of intense investigation (2, 4–6).STEP₆₁ (striatal-enriched protein tyrosine phosphatase) is a brain-specific phosphatase enriched in the striatum and in other regions, including cortex, hippocampus, and substantia nigra (7–9). STEP₆₁ levels are elevated in several disorders, including Alzheimer’s disease, schizophrenia, and fragile X syndrome (10–12). STEP₆₁ levels are normally regulated by the ubiquitin proteasome system, and disruption of this pathway leads to an accumulation of STEP₆₁ in both Alzheimer’s disease and schizophrenia (10, 11).Substrates of STEP₆₁ include ERK1/2, Pyk2, Fyn, the GluN2B subunit of the NMDA receptor, and the GluA2 subunit of the AMPA receptor. The current model of STEP₆₁ function is that it opposes the development of synaptic strengthening by dephosphorylating regulatory tyrosines on these substrates. In the case of the kinases, STEP₆₁-mediated dephosphorylation of the regulatory Tyr within the activation loop inactivates these enzymes (13–16). STEP-mediated dephosphorylation of Tyr residues in the glutamate receptor subunits results in internalization of GluN1/GluN2B and GluA1/GluA2 receptor complexes (17–20). As a result, STEP KO mice have an increase in the basal Tyr phosphorylation of its substrates, including ERK1/2 and its downstream target pCREB (21, 22).Overexpression of STEP disrupts synaptic function, and thereby contributes to cognitive and behavioral deficits (23). Consistent with this hypothesis, genetic or pharmacologic reduction of STEP activity in several disorders in which STEP levels are elevated reverses the biochemical and cognitive deficits that are present (19, 24), and STEP KO mice demonstrate enhanced hippocampal long-term potentiation and enhanced hippocampal- and amygdalar-dependent memory tasks (22, 25).Direct mutations of the E3 ligase parkin (PARK2) result in autosomal recessive juvenile parkinsonism (AR-JP), with early onset of PD symptoms (26, 27); disruption of parkin activity is also implicated in sporadic PD (28–30). Moreover, PD toxins such as MPTP, rotenone, paraquat, and 6-hydroxydopamine alter parkin levels or its ligase activity and result in the accumulation of parkin substrates (31–35). Identification of new parkin substrates and characterization of their role or roles in synaptic function should result in a better understanding the molecular basis of PD.Here we identify parkin as an E3 ligase that ubiquitinates STEP₆₁. STEP₆₁ levels are increased in human PD brains and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD models and are associated with a decrease in the phosphorylation of ERK1/2 and CREB. As an increase in STEP₆₁ expression disrupts synaptic function and contributes to the cognitive deficits in several disorders, these findings suggest that the increase in STEP₆₁ levels in PD may contribute to the pathophysiology of this disorder.     

Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

In type-2 diabetes (T2D) and Parkinson’s disease (PD), polypeptide assembly into amyloid fibers plays central roles: in PD, α-synuclein (aS) forms amyloids and in T2D, amylin [islet amyloid polypeptide (IAPP)] forms amyloids. Using a combination of biophysical methods in vitro we have investigated whether aS, IAPP, and unprocessed IAPP, pro-IAPP, polypeptides can cross-react. Whereas IAPP forms amyloids within minutes, aS takes many hours to assemble into amyloids and pro-IAPP aggregates even slower under the same conditions. We discovered that preformed amyloids of pro-IAPP inhibit, whereas IAPP amyloids promote, aS amyloid formation. Amyloids of aS promote pro-IAPP amyloid formation, whereas they inhibit IAPP amyloid formation. In contrast, mixing of IAPP and aS monomers results in coaggregation that is faster than either protein alone; moreover, pro-IAPP can incorporate aS monomers into its amyloid fibers. From this intricate network of cross-reactivity, it is clear that the presence of IAPP can accelerate aS amyloid formation. This observation may explain why T2D patients are susceptible to developing PD.Parkinson’s disease (PD) is the second most common neurological disorder and the most common movement disorder. It is characterized by widespread degeneration of subcortical structures of the brain, especially dopaminergic neurons in the substantia nigra. These changes are coupled with bradykinesia, rigidity, and tremor, resulting in difficulties in walking and abnormal gait in patients (1). The assembly process of the intrinsically unstructured 140-residue protein α-synuclein (aS) into amyloid fibers has been linked to the molecular basis of PD. aS is a major component of amyloid aggregates found in Lewy body inclusions, which are the pathological hallmark of PD, and duplications, triplications, and point mutations in the aS gene are related to familial PD cases (2, 3). The exact function of aS is unknown, but it is suggested to be involved in synaptic vesicle release and trafficking, regulation of enzymes and transporters, and control of the neuronal apoptotic response (4, 5). aS is present at presynaptic nerve terminals (6–8) and, intriguingly, also in many cells outside the brain (e.g., red blood cells and pancreatic β-cells). aS can assemble via oligomeric intermediates to amyloid fibrils under pathological conditions (9). Although soluble aS oligomers have been proposed to be toxic (10, 11), work with preformed aS fibrils has demonstrated that the amyloid fibrils themselves are toxic and can be transmitted from cell to cell and are also able to cross the blood–brain barrier (12–14).Type-2 diabetes (T2D) is another disease involving amyloid formation. Here, the primary pathological characteristic is islet amyloid of the hormone amylin, also known as islet amyloid polypeptide (IAPP), in pancreatic β-cells (15–18). The process of islet amyloid formation (19–21) leads to pancreatic β-cell dysfunction, cell death, and development of diabetes. IAPP (37 residues, natively unfolded) is cosecreted with insulin after enzymatic maturation of prohormones pro-IAPP (67 residues) and proinsulin in secretory granules. IAPP and insulin play roles in controlling gastric emptying, glucose homeostasis, and in the suppression of glucagon release. Although not understood on a mechanistic level, impairment of prohormone processing has been thought to play a role in initiation and progression of T2D (22, 23). Insulin and pro-IAPP (22, 24–26), but not proinsulin, can inhibit IAPP amyloid formation in vitro and in mice, suggesting that accumulation of unprocessed proinsulin may promote IAPP amyloid formation (22, 24). Insulin-degrading enzyme (IDE) is a conserved metallopeptidase that can degrade insulin and a variety of other small peptides including IAPP in the pancreas (27, 28). Genome-wide association studies have linked IDE to T2D (29, 30) and Ide mutant mice were found to have impaired glucose-stimulated insulin secretion as well as increased levels of IAPP, insulin, and, surprisingly, aS in pancreatic islets (31, 32). Here, aS may be associated with insulin biogenesis and exocytic release, as it was found to localize with insulin-secretory granules in pancreatic β-cells (33). We recently demonstrated in vitro that IDE readily inhibits aS amyloid formation via C-terminal binding and, in parallel, IDE activity toward insulin and other small substrates increases (34, 35).Together, the key role of aS in PD and the inverse correlation of impaired insulin secretion and increased aS levels in the pancreatic β-cells, imply that PD and T2D may be connected. In support, reports have suggested that patients with T2D are predisposed toward PD (36, 37). For Alzheimer’s disease (AD), a direct link with T2D was found (15, 38). Amyloid fiber seeds of the AD peptide, amyloid-β, were shown to efficiently accelerate amyloid formation of IAPP in vitro (39, 40) and IAPP was part of amyloid-β plaque found in mice brains (41). To address the unexplored question of cross-reactivity between the amyloidogenic peptides in PD and T2D, we here investigated cross-reactivity among aS, IAPP, and pro-IAPP using biophysical methods in vitro.     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

A descending dopamine pathway conserved from basal vertebrates to mammals

Dopamine neurons are classically known to modulate locomotion indirectly through ascending projections to the basal ganglia that project down to brainstem locomotor networks. Their loss in Parkinson’s disease is devastating. In lampreys, we recently showed that brainstem networks also receive direct descending dopaminergic inputs that potentiate locomotor output. Here, we provide evidence that this descending dopaminergic pathway is conserved to higher vertebrates, including mammals. In salamanders, dopamine neurons projecting to the striatum or brainstem locomotor networks were partly intermingled. Stimulation of the dopaminergic region evoked dopamine release in brainstem locomotor networks and concurrent reticulospinal activity. In rats, some dopamine neurons projecting to the striatum also innervated the pedunculopontine nucleus, a known locomotor center, and stimulation of the dopaminergic region evoked pedunculopontine dopamine release in vivo. Finally, we found dopaminergic fibers in the human pedunculopontine nucleus. The conservation of a descending dopaminergic pathway across vertebrates warrants re-evaluating dopamine’s role in locomotion.Dopaminergic neurons represent a vital neuromodulatory component essential for vertebrate motor control, and their loss in neurodegenerative disease is devastating. The meso-diencephalic dopamine (DA) neurons are known to provide ascending projections to the basal ganglia, which, in turn, provide input to cortical structure in mammals but also project caudally to the mesencephalic locomotor region (MLR), a highly conserved structure that controls locomotion in all vertebrates investigated to date (1–7; for review, see ref. 8). A growing body of evidence supports the view that basal ganglia connectivity is highly conserved among vertebrates, from lampreys to mammals (9–11; for review, see ref. 12), with some interspecies differences recently highlighted (13). As such, the homology between DA cell populations remains to be resolved in vertebrates. As a general rule, DA neurons from the meso-diencephalon send projections to the striatum in all vertebrates. In lampreys and teleosts, those neurons are located only in the diencephalon (posterior tuberculum), but in tetrapods and cartilaginous fishes (14) they are located in both the diencephalon and the mesencephalon. An increasing number of authors seem to agree with the hypothesis that at least some of the meso-diencephalic DA neurons located in the diencephalon are homologous in all vertebrates, and thus, homologous to at least a portion of the mammalian substantia nigra pars compacta (SNc)/ventral tegmental area (VTA) (13, 15–19; for review, see ref. 20). Alternatively, it was suggested that the posterior tuberculum DA neurons projecting to the striatum in zebrafish are homologs of the mammalian DA neurons of the A11 group (21). This will be discussed below in light of the results of the present study.In lampreys, only a few meso-diencephalic DA neurons send ascending projections to the striatum (9, 22); the majority of DA neurons send a direct descending projection to the MLR (22, 23), where DA is released and increases locomotor output through D1 receptors (22). These results demonstrate that the descending dopaminergic pathway to the MLR is an important modulator of locomotor output, but it remains to be determined whether this pathway is conserved in higher vertebrates.The existence of a descending dopaminergic pathway that powerfully increases locomotor output has important implications for Parkinson’s disease, which involves the meso-diencephalic DA neurons. A loss of descending dopaminergic projections could play a role in the locomotor deficits systematically observed in that disease. Because of the highly conserved nature of both the dopaminergic system and brainstem locomotor circuitry in vertebrates, we hypothesized that a direct descending dopaminergic pathway to the MLR also exists in higher vertebrates. Previous anatomical (24, 25) and electrophysiological (26) studies in rats support the idea of a descending connection from the SNc to the pedunculopontine nucleus [PPN, considered part of the MLR (2)]. Moreover, dopaminergic terminals were found in the PPN of monkeys (27), but the origin of this projection is still unknown in mammals.Here, we investigated whether the direct descending projection from meso-diencephalic DA neurons to the MLR is present in two tetrapods, the salamander and the rat. Moreover, we supplement our analyses with anatomical data from human brain tissue. Using traditional and virogenetic axonal tracing, immunofluorescence, in vivo voltammetry, and calcium imaging of reticulospinal neurons, we provide anatomical and functional evidence strongly supporting a conserved role for the descending projections of meso-diencephalic DA neurons in the regulation of brainstem locomotor networks across the vertebrate subphylum.     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

Fog and rain in the Amazon

The diurnal and seasonal water cycles in the Amazon remain poorly simulated in general circulation models, exhibiting peak evapotranspiration in the wrong season and rain too early in the day. We show that those biases are not present in cloud-resolving simulations with parameterized large-scale circulation. The difference is attributed to the representation of the morning fog layer, and to more accurate characterization of convection and its coupling with large-scale circulation. The morning fog layer, present in the wet season but absent in the dry season, dramatically increases cloud albedo, which reduces evapotranspiration through its modulation of the surface energy budget. These results highlight the importance of the coupling between the energy and hydrological cycles and the key role of cloud albedo feedback for climates over tropical continents.Tropical forests, and the Amazon in particular, are the biggest terrestrial CO₂ sinks on the planet, accounting for about 30% of the total net primary productivity in terrestrial ecosystems. Hence, the climate of the Amazon is of particular importance for the fate of global CO₂ concentration in the atmosphere (1). Besides the difficulty of estimating carbon pools (1–3), our incapacity to correctly predict CO₂ fluxes in the continental tropics largely results from inaccurate simulation of the tropical climate (1, 2, 4, 5). More frequent and more intense droughts in particular are expected to affect the future health of the Amazon and its capacity to act as a major carbon sink (6–8). The land surface is not isolated, however, but interacts with the weather and climate through a series of land−atmosphere feedback loops, which couple the energy, carbon, and water cycles through stomata regulation and boundary layer mediation (9).Current General Circulation Models (GCMs) fail to correctly represent some of the key features of the Amazon climate. In particular, they (i) underestimate the precipitation in the region (10, 11), (ii) do not reproduce the seasonality of either precipitation (10, 11) or surface fluxes such as evapotranspiration (12), and (iii) produce errors in the diurnal cycle and intensity of precipitation, with a tendency to rain too little and too early in the day (13, 14). In the more humid Western part of the basin, surface incoming radiation, evapotranspiration, and photosynthesis all tend to peak in the dry season (15–17), whereas GCMs simulate peaks of those fluxes in the wet season (10, 11). Those issues might be related to the representation of convection (1, 2, 4, 5, 13, 14) and vegetation water stress (6–8, 15–17) in GCMs.We here show that we can represent the Amazonian climate using a strategy opposite to GCMs in which we resolve convection and parameterize the large-scale circulation (Methods). The simulations lack many of the biases observed in GCMs and more accurately capture the differences between the dry and wet season of the Amazon in surface heat fluxes and precipitation. Besides top-of-the-atmosphere insolation, the simulations require the monthly mean temperature profile as an input. We demonstrate that this profile, whose seasonal cycle itself is a product of the coupled ocean−land−atmosphere dynamics, mediates the seasonality of the Amazonian climate by modulating the vertical structure of the large-scale circulation in such a way that thermal energy is less effectively ventilated in the rainy season.     

Accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in Huntington’s disease

Mitochondrial dysfunction is found in the brain and peripheral tissues of patients diagnosed with Huntington’s disease (HD), an irreversible neurodegenerative disease of which aging is a major risk factor. Mitochondrial function is encoded by not only nuclear DNA but also DNA within mitochondria (mtDNA). Expansion of mtDNA heteroplasmies (coexistence of mutated and wild-type mtDNA) can contribute to age-related decline of mitochondrial function but has not been systematically investigated in HD. Here, by using a sensitive mtDNA-targeted sequencing method, we studied mtDNA heteroplasmies in lymphoblasts and longitudinal blood samples of HD patients. We found a significant increase in the fraction of mtDNA heteroplasmies with predicted pathogenicity in lymphoblasts from 1,549 HD patients relative to lymphoblasts from 182 healthy individuals. The increased fraction of pathogenic mtDNA heteroplasmies in HD lymphoblasts also correlated with advancing HD stages and worsened disease severity measured by HD motor function, cognitive function, and functional capacity. Of note, elongated CAG repeats in HTT promoted age-dependent expansion of pathogenic mtDNA heteroplasmies in HD lymphoblasts. We then confirmed in longitudinal blood samples of 169 HD patients that expansion of pathogenic mtDNA heteroplasmies was correlated with decline in functional capacity and exacerbation of HD motor and cognitive functions during a median follow-up of 6 y. The results of our study indicate accelerated decline of mtDNA quality in HD, and highlight monitoring mtDNA heteroplasmies longitudinally as a way to investigate the progressive decline of mitochondrial function in aging and age-related diseases.

Huntington’s disease (HD) is a monogenic disorder caused by the expansion of cytosine–adenine–guanine trinucleotide (CAG) repeats in the HTT gene at chromosome 4p16.3 (1). Although HTT is expressed in various tissues, the brain, particularly the striatum, is vulnerable to mutant huntingtin (mHTT)-associated toxicity (2). The average age at onset of the characteristic motor symptoms of HD is between 40 and 50 y old, followed by a progressive decline of motor, cognitive, and psychiatric functions for an average of 20 y prior to death (3).The biological processes that determine the onset and progression of HD are still elusive. Recent studies suggest that mitochondrial dysfunction may be involved in HD pathogenesis (4, 5). Mitochondria are subcellular organelles of eukaryotes, which play vital roles in maintaining energetic and metabolic homeostasis (6, 7). Evidence for mitochondrial dysfunction in HD was first reported in the postmortem brain of HD patients, which show low mitochondrial oxidative phosphorylation (OXPHOS) protein activity and energy deficits (8–10). Mitochondrial dysfunction was further found in peripheral tissues and cell lines of HD patients, such as blood, lymphoblasts, skeletal muscle, and skin fibroblasts (11–17).Several molecular mechanisms have been proposed to connect mHTT to mitochondrial dysfunction. Studies in HD knockin mice indicate that toxic fragments derived from mHTT can suppress mitochondrial biogenesis and energy metabolism (18). mHTT has also been found to physically interact with mitochondria, reducing mitochondrial membrane potential (13, 19). Furthermore, mHTT may stimulate mitochondrial network fragmentation (20–22), and it has recently been found to impair mitophagy (23–28), an evolutionarily conserved quality control system in eukaryotes to selectively remove dysfunctional mitochondria (29). Perturbation of mitochondrial tubular networks, morphology, and mitophagy are pathological features common to various neurodegenerative diseases (30, 31).Mitochondrial function is determined not only by the nuclear genome but also by the mitochondrial genome (mtDNA). Human mtDNA is a 16.6-kb circular DNA located within mitochondria. It encodes 13 evolutionarily conserved proteins in four of the five OXPHOS protein complexes (32). The accumulation of mtDNA mutations in somatic tissues has been suggested as a possible driver of age-related mitochondrial dysfunction (33). Transgenic mice with an increased level of mtDNA mutations manifest progeroid phenotypes and early neurodegeneration that resemble human aging (34, 35). Clonal expansion of preexisting mtDNA mutations in somatic tissues has also been shown to contribute to accelerated mitochondrial aging and OXPHOS defects in human diseases (36, 37).Because there are multiple copies of mtDNA in a single cell, mutations can arise and coexist with wild-type mtDNA in a state called heteroplasmy, which has been linked to a variety of mitochondrial disorders in humans (32, 38). Our previous study on lymphoblasts from the 1,000 Genomes project indicates that about 90% of individuals in the general population carry at least one heteroplasmy in mtDNA, and purifying selection keeps most pathogenic heteroplasmies at a low fraction (39). Thus, when such a selective constraint on mitochondria is weakened under certain conditions (40), such as the presence of mHTT (20–28), these low-fraction pathogenic heteroplasmies may increase in their fractions in cells, culminating in dysfunctional mitochondria and related energy deficits (32).In the current study, we hypothesized that HD progression is partially driven by the deterioration of mtDNA quality. Since HTT is universally expressed, and mitochondrial dysfunction has been repeatedly observed in peripheral tissues (11–16), we surmised that HD-associated mtDNA changes can be detected in peripheral tissues and cell lines of HD patients, such as blood-derived lymphoblasts, which are readily available in large patient cohorts and thus can provide increased power for identifying mtDNA changes in HD. To test this hypothesis, we employed a sensitive mtDNA targeted sequencing approach, STAMP (sequencing by targeted amplification of multiplex probes) (41), to assess mtDNA heteroplasmies in lymphoblast and longitudinal blood samples from HD patients and healthy control individuals in the European Huntington’s Disease Network’s REGISTRY study (hereafter referred to as REGISTRY) (42). We achieved ultradeep sequencing coverage on mtDNA in these samples and revealed an accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in HD, illustrating a molecular feature underlying HD biology.     

Repeat sequences limit the effectiveness of lateral gene transfer and favored the evolution of meiotic sex in early eukaryotes

The transition from prokaryotic lateral gene transfer to eukaryotic meiotic sex is poorly understood. Phylogenetic evidence suggests that it was tightly linked to eukaryogenesis, which involved an unprecedented rise in both genome size and the density of genetic repeats. Expansion of genome size raised the severity of Muller’s ratchet, while limiting the effectiveness of lateral gene transfer (LGT) at purging deleterious mutations. In principle, an increase in recombination length combined with higher rates of LGT could solve this problem. Here, we show using a computational model that this solution fails in the presence of genetic repeats prevalent in early eukaryotes. The model demonstrates that dispersed repeat sequences allow ectopic recombination, which leads to the loss of genetic information and curtails the capacity of LGT to prevent mutation accumulation. Increasing recombination length in the presence of repeat sequences exacerbates the problem. Mutational decay can only be resisted with homology along extended sequences of DNA. We conclude that the transition to homologous pairing along linear chromosomes was a key innovation in meiotic sex, which was instrumental in the expansion of eukaryotic genomes and morphological complexity.

The genes for meiosis are universal among eukaryotes, indicating that sex evolved before the divergence of the first eukaryotic clades (1, 2). It evolved from the molecular machinery for lateral gene transfer (LGT), which facilitates genetic exchange in archaea and bacteria (1, 3, 4). Prokaryotes possess homologs of the canonical molecular machinery for meiotic sex, including proteins of the SMC gene family of adenosine triphosphatases necessary for chromosome cohesion and condensation (5), as well as actin and tubulin, required for daughter cell separation and the movement of chromosomes (6). The Rad51/Dcm1 gene family, which plays a central role in meiosis, also has high protein sequence similarity with RecA, responsible for homologous search and recombination in prokaryotes (7, 8). But why eukaryotes requisitioned this existing molecular machinery to evolve a completely new mechanism of reproduction, inheritance, and genetic exchange—meiotic sex—remains obscure.Transformation is one of the major routes of genetic exchange via LGT in bacteria and involves the acquisition of environmental DNA (eDNA), followed by recombination into the host genome (8, 9). By allowing genetic exchange between lineages, transformation can restore genes that have been disrupted through mutation or deletion (10–12), counter the effects of genetic drift and reverse Muller’s ratchet (11, 13), and accelerate adaptation by reducing selective interference (14, 15). Previous modeling work has shown that the expansion of early eukaryote genome size was likely to have caused the failure of LGT (13). While LGT via transformation helps to purge deleterious mutations (11), this benefit rapidly wanes as genome size increases because of the difficulty of matching individual mutations with eDNA (13). LGT can resist mutation accumulation in larger genomes by combining more frequent recombination with increased recombination length, the mean length of DNA picked up from the environment and recombined into the host cell genome (13). But the distribution of recombination length in bacteria is skewed toward short eDNA sequences, with a median length that encompasses at most just a few genes (16–18). In addition, bacteria typically cleave eDNA, shortening recombination length. While there are constraints on the rate of uptake and recombination through limited eDNA availability and sequence homology (12, 18), prokaryotes plainly did not follow the eukaryotic trajectory toward recombination across whole chromosomes.After the endosymbiotic event that gave rise to the first eukaryotes, the archaeal host’s genome greatly expanded with genes of bacterial endosymbiotic origin and through gene duplication and divergence, which enabled a range of novel functions (19, 20). This is estimated to have doubled gene number in the last eukaryotic common ancestor (LECA) (21, 22). The extra energetic availability provided by the protomitochondrial endosymbiont released bioenergetic constraints over prokaryotic cell and genome size (23, 24). But this came with the cost of maintaining a larger genome (19, 21, 24, 25). Early eukaryote genome size expansion also reflected an increase in the density of repeat sequences, arising from gene duplication and the spread of mobile genetic elements (25, 26). Mobile retroelements of endosymbiotic origin are thought to have spread widely through the protoeukaryote host genome, leading to a proliferation of self-splicing introns (27–29). These selfish elements are present in many bacterial species, almost always at low copy numbers (<10 per genome) (30), but likely increased in a more uninhibited manner, perhaps exploiting the nonhomologous end-joining mechanism of DNA repair found throughout eukaryotes (31). Novel intron density is thought to have reached a density comparable to that seen in modern eukaryote species (29).The need to restrict ectopic recombination caused by increased repeat density might have played a pivotal role in determining the evolution of meiosis (32). However, the possible involvement of such repeat sequences has not been investigated in previous quantitative models of LGT or the transition to meiotic sex (10, 11, 13). In prokaryotes, high repeat density is associated with a high probability of ectopic recombination, increasing the rates of deletions, insertions, and other genomic rearrangements (33, 34). Recombination errors caused by the presence of repeat sequences introduce an additional cost to LGT and potentially constrain the benefits of increased recombination length and LGT frequency. Here, we investigate whether the sharp increase in repeat density in early eukaryotes could have forced them to abandon LGT in favor of meiosis. To investigate this hypothesis, we develop a computational model of mutation and selection in a population undergoing LGT via transformation in the presence of genetic repeats. The model highlights a tradeoff between the benefits of LGT (greater genetic variance, enhancing purifying selection) and its cost (loss of genetic information through ectopic recombination). This leads to the view that the transition to meiotic sex was driven by the need for purifying selection in the expanding and repeat-rich genomes of early eukaryotes, which could not be met by increases in recombination length or LGT rate.     

From the Cover: Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.Genetics contribute to the pathogenesis of Parkinson disease (PD) in two ways. Mutations in several genes can cause inherited PD (1), and risk factor variants contribute to the risk of sporadic PD (2). Some genes contribute to both mechanisms. These pleomorphic risk loci (3) include genes that encode α-synuclein and leucine-rich repeat kinase 2 (LRRK2) (4). However, risk factors for sporadic PD identified by genome-wide association studies (GWASs) (5–9) actually nominate large genomic loci with multiple candidate genes (10). These loci may include variants that change amino acids or affect disease risk through gene expression (11). Also, whether all of the genes for PD converge on a small number of biological pathways is unknown (1). It is, therefore, important to develop unbiased approaches that would resolve whether genes for PD have similar biological functions and understand the mechanism(s) of disease risk. Here, we examine one genetic cause of PD (LRRK2) and show that identifying protein interaction partners can clarify disease mechanisms.     

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

A lipid switch unlocks Parkinson’s disease-associated ATP13A2

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn²⁺, and Zn²⁺ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders.Neuronal fitness depends on optimal lysosomal function and efficient lysosomal delivery of proteins and organelles by autophagy for subsequent breakdown (1, 2). Kufor–Rakeb syndrome (KRS) is an autosomal recessive form of Parkinson’s disease (PD) associated with dementia, which is caused by mutations in ATP13A2/PARK9 (3). Mutations in or knockdown (KD) of ATP13A2 lead to lysosomal dysfunctions, including reduced lysosomal acidification, decreased degradation of lysosomal substrates (4), impaired autophagosomal flux (4, 5), and accumulation of fragmented mitochondria (5, 6). By contrast, overexpression (OE) of Ypk9p (i.e., the yeast ATP13A2 ortholog) protects yeast against toxicity of α-synuclein (7), which is the major protein in Lewy bodies, the abnormal protein aggregates that develop inside nerve cells in PD. This protective effect of ATP13A2 on α-synuclein toxicity is conserved in yeast, Caenorhabditis elegans, and rat neuronal cells (7). Because ATP13A2 imparts resistance to Mn²⁺ (7–9) and Zn²⁺ (10–12), it was proposed that ATP13A2 may function as a Mn²⁺ (7–9) and/or Zn²⁺ transporter (10–12).ATP13A2 belongs to the P5 subfamily of the P-type ATPase superfamily, which comprises five subfamilies (P1–5) of membrane transporters. P-type ATPases hydrolyze ATP to actively transport inorganic ions across membranes or lipids between membrane leaflets (reviewed in ref. 13). During the transport cycle, a phospho-intermediate is formed on a conserved aspartate residue (14). The human P5-type ATPases are divided into two groups, P5A (ATP13A1) and P5B (ATP13A2–5), but their transport specificity has not been established (14–16).P-type ATPases comprise a membrane-embedded core of six transmembrane (TM) helices (M1–6) that form the substrate binding site(s) and entrance/exit pathways for the transported substrate (13). Whereas four extra C-terminal TM helices (M7–10) are common, additional N-terminal TM helices are only observed in the P1B heavy metal pumps (17). Topology predictions indicate that ATP13A2 and other P5 members also contain additional N-terminal TM helices, which might serve a subclass-specific function (14, 15). Here, we demonstrate that the ATP13A2 N terminus is a critical regulatory element involved in lipid binding and ATP13A2 activation, providing protection to mitochondrial stress in a cellular PD model.     

Spontaneous nucleation and fast aggregate-dependent proliferation of α-synuclein aggregates within liquid condensates at neutral pH

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson’s disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.

Parkinson’s disease is the most common neurodegenerative movement disorder (1, 2). A distinctive pathophysiological signature of this disease is the presence of abnormal intraneuronal protein deposits known as Lewy bodies (3, 4). One of the main components of Lewy bodies is α-synuclein (5), a peripheral membrane protein highly abundant at neuronal synapses (6, 7) and genetically linked with Parkinson’s disease (8, 9). This 140-residue disordered protein can be subdivided into three domains, an amphipathic N-terminal region (amino acids 1 to 60), a central hydrophobic region (non-amyloid-β component, or NAC, amino acids 61 to 95), and an acidic proline-rich C-terminal tail (amino acids 96 to 140) (7). Although α-synuclein aggregation is characteristic of Parkinson’s disease and related synucleinopathies, the corresponding mechanism and its possible pathological role in disease are not yet fully understood.Generally, the aggregation process of proteins proceeds through a series of interconnected microscopic steps, including primary nucleation, elongation, and secondary nucleation (10, 11). During primary nucleation, the self-assembly of proteins from their native, monomeric form leads to the formation of oligomeric species, an event that may occur in solution or on surfaces including biological membranes (12, 13). The formation of these oligomers is typically a slow event governed by high kinetic barriers (10, 11). Once formed, the oligomers may convert into ordered assemblies rich in β structure, which are capable of further growth into fibrillar aggregates (14). In many cases, the surfaces of existing fibrillar aggregates then further catalyze the formation of new oligomers (15, 16). This secondary nucleation process is typically characterized by the assembly of protein monomers on the surface of fibrils that eventually nucleate into new oligomeric species (15, 16). This autocatalytic mechanism generates rapid fibril proliferation (15).In the case of the aggregation process of α-synuclein, several key questions are still open, including two that we are addressing in this study. The first concerns whether there are cellular conditions under which α-synuclein can undergo spontaneous aggregation, and the second whether the proliferation of α-synuclein fibrils by aggregate-dependent feedback processes can take place at physiological pH. These questions are relevant because according to our current knowledge, α-synuclein aggregation does not readily take place spontaneously in the absence of contributing factors such as lipid membranes. Furthermore, secondary nucleation contributes significantly to the aggregation process only at acidic pH (13, 17). It thus remains challenging to rationalize the links between α-synuclein aggregation and Parkinson’s disease.To address this problem, we investigated whether it is possible to leverage the recent finding that α-synuclein can undergo a phase separation process resulting in the formation of dense liquid condensates (18–21). Phase separation has recently emerged as a general phenomenon associated with a wide variety of cellular functions (22–25) and closely linked with human disease (23, 26–29). This process has been reported for a wide range of proteins implicated in neurodegenerative conditions, including tau, fused in sarcoma (FUS), and TAR DNA binding protein 43 (TDP-43) (30–32). Since it has also been shown that protein aggregation can take place within liquid condensates (19, 26, 32–36), we asked whether it is possible to characterize at the microscopic level the condensate-induced aggregation mechanism of α-synuclein by determining the kinetic rate constants of the corresponding microscopic processes.To enable the accurate determination of the rate constants for the microscopic steps in α-synuclein aggregation within condensates, we developed fluorescence-based aggregation assays to monitor both the spontaneous aggregation of α-synuclein and the aggregation in the presence of aggregate seeds. Using these assays within the framework of a kinetic theory of protein aggregation (10, 11, 37), we show that α-synuclein can undergo spontaneous homogenous primary nucleation and fast aggregate-dependent proliferation within condensates at physiological pH.