Ariadna Recasens

Pathogenic Lysosomal Depletion in Parkinson's Disease

Mounting evidence suggests a role for autophagy dysregulation in Parkinsons disease (PD). The bulk degradation of cytoplasmic proteins (including α-synuclein) and organelles (such as mitochondria) is mediated by macroautophagy, which involves the sequestration of cytosolic components into autophagosomes (AP) and its delivery to lysosomes. Accumulation of AP occurs in postmortem brain samples from PD patients, which has been widely attributed to an induction of autophagy. However, the cause and pathogenic significance of these changes remain unknown. Here we found in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD that AP accumulation and dopaminergic cell death are preceded by a marked decrease in the amount of lysosomes within dopaminergic neurons. Lysosomal depletion was secondary to the abnormal permeabilization of lysosomal membranes induced by increased mitochondrial-derived reactive oxygen species. Lysosomal permeabilization resulted in a defective clearance and subsequent accumulation of undegraded AP and contributed directly to neurodegeneration by the ectopic release of lysosomal proteases into the cytosol. Lysosomal breakdown and AP accumulation also occurred in PD brain samples, where Lewy bodies were strongly immunoreactive for AP markers. Induction of lysosomal biogenesis by genetic or pharmacological activation of lysosomal transcription factor EB restored lysosomal levels, increased AP clearance and attenuated 1-methyl-4-phenylpyridinium-induced cell death. Similarly, the autophagy-enhancer compound rapamycin attenuated PD-related dopaminergic neurodegeneration, both in vitro and in vivo, by restoring lysosomal levels. Our results indicate that AP accumulation in PD results from defective lysosomal-mediated AP clearance secondary to lysosomal depletion. Restoration of lysosomal levels and function may thus represent a novel neuroprotective strategy in PD.

Lewy body extracts from Parkinson disease brains trigger α‐synuclein pathology and neurodegeneration in mice and monkeys

Mounting evidence suggests that α‐synuclein, a major protein component of Lewy bodies (LB), may be responsible for initiating and spreading the pathological process in Parkinson disease (PD). Supporting this concept, intracerebral inoculation of synthetic recombinant α‐synuclein fibrils can trigger α‐synuclein pathology in mice. However, it remains uncertain whether the pathogenic effects of recombinant synthetic α‐synuclein may apply to PD‐linked pathological α‐synuclein and occur in species closer to humans.

Alpha-synuclein spreading in Parkinson’s disease

Formation and accumulation of misfolded protein aggregates are a central hallmark of several neurodegenerative diseases. In Parkinson’s disease (PD), the aggregation-prone protein alpha-synuclein (α-syn) is the culprit. In the past few years, another piece of the puzzle has been added with data suggesting that α-syn may self-propagate, thereby contributing to the progression and extension of PD. Of particular importance, it was the seminal observation of Lewy bodies (LB), a histopathological signature of PD, in grafted fetal dopaminergic neurons in the striatum of PD patients. Consequently, these findings were a conceptual breakthrough, generating the “host to graft transmission” hypothesis, also called the “prion-like hypothesis.” Several in vitro and in vivo studies suggest that α-syn can undergo a toxic templated conformational change, spread from cell to cell and from region to region, and initiate the formation of “LB–like aggregates,” contributing to the PD pathogenesis. Here, we will review and discuss the current knowledge for such a putative mechanism on the prion-like nature of α-syn, and discuss about the proper use of the term prion-like.

Optic atrophy 1 mediates mitochondria remodeling and dopaminergic neurodegeneration linked to complex I deficiency

Mitochondrial complex I dysfunction has long been associated with Parkinson’s disease (PD). Recent evidence suggests that mitochondrial involvement in PD may extend beyond a sole respiratory deficit and also include perturbations in mitochondrial fusion/fission or ultrastructure. Whether and how alterations in mitochondrial dynamics may relate to the known complex I defects in PD is unclear. Optic atrophy 1 (OPA1), a dynamin-related GTPase of the inner mitochondrial membrane, participates in mitochondrial fusion and apoptotic mitochondrial cristae remodeling. Here we show that complex I inhibition by parkinsonian neurotoxins leads to an oxidative-dependent disruption of OPA1 oligomeric complexes that normally keep mitochondrial cristae junctions tight. As a consequence, affected mitochondria exhibit major structural abnormalities, including cristae disintegration, loss of matrix density and swelling. These changes are not accompanied by mitochondrial fission but a mobilization of cytochrome c from cristae to intermembrane space, thereby lowering the threshold for activation of mitochondria-dependent apoptosis by cell death agonists in compromised neurons. All these pathogenic changes, including mitochondrial structural remodeling and dopaminergic neurodegeneration, are abrogated by OPA1 overexpression, both in vitro and in vivo. Our results identify OPA1 as molecular link between complex I deficiency and alterations in mitochondrial dynamics machinery and point to OPA1 as a novel therapeutic target for complex I cytopathies, such as PD.

BAX channel activity mediates lysosomal disruption linked to Parkinson disease

Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

Role of microRNAs in the Regulation of α-Synuclein Expression: A Systematic Review

Growing evidence suggests that increased levels of α-synuclein might contribute to the pathogenesis of Parkinson’s disease (PD) and therefore, it is crucial to understand the mechanisms underlying α-synuclein expression. Recently, microRNAs (miRNAs) have emerged as key regulators of gene expression involved in several diseases such as PD and other neurodegenerative disorders. A systematic literature search was performed here to identify microRNAs that directly or indirectly impact in α-synuclein expression/accumulation and describe its mechanism of action. A total of 27 studies were incorporated in the review article showing evidences that six microRNAs directly bind and regulate α-synuclein expression while several miRNAs impact on α-synuclein expression indirectly by targeting other genes. In turn, α-synuclein overexpression also impacts miRNAs expression, indicating the complex network between miRNAs and α-synuclein. From the current knowledge on the central role of α-synuclein in PD pathogenesis/progression, miRNAs are likely to play a crucial role at different stages of PD and might potentially be considered as new PD therapeutic approaches.

Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson’s Disease

Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinsons disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%-40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD.

In vivo models of alpha-synuclein transmission and propagation

The abnormal accumulation of α-synuclein aggregates in neurons, nerve fibers, or glial cells is the hallmark of a group of neurodegenerative diseases known collectively as α-synucleinopathies. Clinical, neuropathological, and experimental evidence strongly suggests that α-synuclein plays a role not only as a trigger of pathological processes at disease inception, but also as a mediator of pathological spreading during disease progression. Specific properties of α-synuclein, such as its ability to pass from one neuron to another, its tendency to aggregate, and its potential to generate self-propagating species, have been described and elucidated in animal models and may contribute to the relentless exacerbation of Parkinson’s disease pathology in patients. Animal models used for studying α-synuclein accumulation, aggregation, and propagation are mostly based on three approaches: (1) intra-parenchymal inoculations of exogenous α-synuclein (e.g., synthetic α-synuclein fibrils), (2) transgenic mice, and (3) animals (mice or rats) in which α-synuclein overexpression is induced by viral vector injections. Whereas pathological α-synuclein changes are consistently observed in these models, important differences are also found. In particular, pronounced pathology in transgenic mice and viral vector-injected animals does not appear to involve self-propagating α-synuclein species. A critical discussion of these models reveals their strengths and limitations and provides the basis for recommendations concerning their use for future investigations.

Selective suppression of α-Synuclein in monoaminergic neurons of mice by intranasal delivery of targeted small interfering RNA or antisense oligonucleotides: Potential therapy for Parkinson's disease

Background: The use of opioid-based therapies in patients with chronic pain is a challenge in the medical practice because of drug abuse liability. This problem is magnified in the case of patients with a previous history of opioid abuse leading to reduced treatment of pain conditions in this population. Surprisingly, few studies have investigated how chronic pain could alter opioid intake patterns and none of them have focused in an opioid dependent population. In addition, although it is known that opioid reinforcement is mediated through the activation of the mesolimbic dopamine (DA) neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), there is uncertainty about the effects of pain on DA transmission within these brain areas and whether pain-induced effects on DA transmission within the VTA-NAc pathway may impact the reinforcing properties of opioids. Methods: In the present study, we investigated the effects of chronic inflammatory pain on i.v. heroin self-administration under fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement. We selected the complete Freund’s adjuvant (CFA) rat model of inflammation to assess the effect of chronic inflammatory pain on opioid abuse. In addition, we investigated the neurochemical changes induced by chronic inflammatory pain on DA transmission within the mesolimbic pathway by conducting in vivo microdialysis studies. All experimental protocols in animal studies were approved by the Institutional Animal Care and Use Committee at Columbia University. Results: First, to examine whether the injection of CFA could affect the correct performance of the self-administration task we analyzed the effect of CFA-induced chronic inflammation on sucrose intake under a FR2 schedule, 2 and 7 days after saline or CFA injection. Neither CFA nor saline injection altered the number of correct responses vs the number of incorrect responses during sucrose FR2 self-administration sessions, indicating that CFA injection does not affect the correct performance of this task. Next, we studied motivated behavior for heroin self-administration. Seven days following iv cannulation, animals were placed in the operant chamber and trained to self administer sucrose until they could complete three consecutive sessions by self administering 60 sucrose pellets per session. Once the animals acquired the self administration behavior, they were given 2 h access to heroin (50 mg/kg/ infusion) under FR1 schedule. After stable drug intake was obtained (defined as 5 consecutive sessions in which the number of infusions did not vary by more than 15% of the mean value obtained across those sessions), the number of responses required for an infusion was increased to 2 and then to 5 for a total of 6 sessions. Then, all animals underwent an initial PR session, where the number of correct responses increases exponentially with each administration, to measure their basal motivation for heroin (50 mg/kg/infusion). Two additional PR sessions at two different time points (2 and 7 days after CFA or saline injection, respectively) were conducted. Pain decreased the breakpoint (maximum number of responses that the animal completes in order to receive a reward) at the two time points tested after CFA injection for heroin at a dose of 50 mg/kg/infusion. These data indicate that CFA-induced pain impacts motivated behavior for self-administration at this lower dose of heroin. Next, we investigated whether this decrease in the break point induced by pain was dependent on the dose of heroin. Animals were trained under FR1 and FR2 schedules (50 mg/kg/infusion heroin) and 2 days after CFA or saline injection they were placed again in the operant box to generate a ‘within-session’ dose response curve. One of three doses of heroin (50, 100, 200 mg/kg/infusion) was made available for a 1-h time period during the session, with a 15-min resting period between doses. Doses were altered by varying the infusion duration. The presentation of the doses was made in an ascending or descending manner. Both dose-response curves showed a deviation from the expected linear dose-response for heroin, suggesting a possible change in the sensitivity (ie of the reinforcing properties) to heroin in the presence of chronic inflammatory pain. Finally, in order to determine whether these pain-induced altered patterns of opioid self-administration were related to the effects of chronic inflammatory pain on DA transmission within the VTA-NAc pathway we conducted in vivo microdialysis studies in CFAand saline-injected rats. We found that chronic inflammatory pain blunted the effects of a 50 mg i.v. heroin challenge on DA release in the NAc. Conclusions: These data suggest that chronic inflammatory pain may produce a rightward shift in the rewarding properties of heroin in opioid dependent rats, such that higher doses of heroin are needed for reliable rates of selfadministration. In addition, our findings indicate that these effects may be related to an attenuation of DA transmission within the VTA-NAc pathway triggered by the presence of chronic inflammatory pain. Therefore, these data suggest that chronic inflammatory pain induces changes in the VTA-NAc pathway which in turn may facilitate opioid dose escalation in order to maintain the rewarding properties of the drug.

Lack of pathogenic potential of peripheral α-synuclein aggregates from Parkinson’s disease patients

In Parkinson’s disease (PD) there is widespread accumulation in the brain of abnormal α-synuclein aggregates forming intraneuronal Lewy bodies (LB). It is now well established that LB-type α-synuclein aggregates also occur in the peripheral autonomic nervous system in PD, from where it has been speculated they may progressively spread to the central nervous system through synaptically-connected brain networks and reach the substantia nigra to trigger herein dopaminergic dysfunction/degeneration and subsequent parkinsonism. Supporting a pathogenic role for α-synuclein aggregates we have previously shown that LB purified from postmortem PD brains promote α-synuclein pathology and dopaminergic neurodegeneration when intracerebrally inoculated into wild-type mice. However, the pathogenic capacity of PD-derived peripheral α-synuclein aggregates remains unknown. Here we addressed this question using purified LB-type α-synuclein aggregates from postmortem PD stellate ganglia (SG), a paravertebral sympathetic ganglion that exhibits consistent and conspicuous Lewy pathology in all PD patients. In contrast to our previous findings using nigral LB extracts, intracerebral inoculation of SG-derived LB into mice did not trigger long-term nigrostriatal neurodegeneration nor α-synuclein pathology. The differential pathogenic capacities of central- and peripheral-derived α-synuclein aggregates appear independent of the absolute amount and basic biochemical properties of α-synuclein within these aggregates and may rely instead on differences in α-synuclein conformation and/or yet unrecognized brain region-specific intrinsic factors. Our results argue against a putative pathogenic capacity of peripheral α-synuclein aggregates to promote α-synuclein pathology in the brain, propagate between neuronal networks or induce neurodegeneration.