Aged and Diseased Neurons Get Lost in Transport.

The maintenance of nucleocytoplasmic compartmentalization is essential for proper cellular function. Recent studies from the Gage and the Hipp labs report impairments in transport across the nuclear envelope in models of normal and pathological neuronal senescence, providing a mechanistic link between cerebral aging and neurodegenerative diseases.

Targeting α-synuclein: Therapeutic options.

The discovery of the central role of α-synuclein (αSyn) in the pathogenesis of Parkinson's disease (PD) has powered, in the last decade, the emergence of novel relevant models of this condition based on viral vector-mediated expression of the disease-causing protein or inoculation of toxic species of αSyn. Although the development of these powerful tools and models has provided considerable insights into the mechanisms underlying neurodegeneration in PD, it has also been translated into the expansion of the landscape of preclinical therapeutic strategies. Much attention is now brought to the proteotoxic mechanisms induced by αSyn and how to block them using strategies inspired by intrinsic cellular pathways such as the enhancement of cellular clearance by the lysosomal-autophagic system, through proteasome-mediated degradation or through immunization. The important effort undertaken by several laboratories and consortia to tackle these issues and identify novel targets warrants great promise for the discovery not only of neuroprotective approaches but also of restorative strategies for PD and other synucleinopathies. In this viewpoint, we summarize the latest advances in this new area of PD research and will discuss promising approaches and ongoing challenges. © 2016 International Parkinson and Movement Disorder Society.

Alpha-Synuclein Produces Early Behavioral Alterations via Striatal Cholinergic Synaptic Dysfunction by Interacting With GluN2D N-Methyl-D-Aspartate Receptor Subunit.

BACKGROUND: Advanced Parkinson's disease (PD) is characterized by massive degeneration of nigral dopaminergic neurons, dramatic motor and cognitive alterations, and presence of nigral Lewy bodies, whose main constituent is α-synuclein (α-syn). However, the synaptic mechanisms underlying behavioral and motor effects induced by early selective overexpression of nigral α-syn are still a matter of debate. METHODS: We performed behavioral, molecular, and immunohistochemical analyses in two transgenic models of PD, mice transgenic for truncated human α-synuclein 1-120 and rats injected with the adeno-associated viral vector carrying wild-type human α-synuclein. We also investigated striatal synaptic plasticity by electrophysiological recordings from spiny projection neurons and cholinergic interneurons. RESULTS: We found that overexpression of truncated or wild-type human α-syn causes partial reduction of striatal dopamine levels and selectively blocks the induction of long-term potentiation in striatal cholinergic interneurons, producing early memory and motor alterations. These effects were dependent on α-syn modulation of the GluN2D-expressing N-methyl-D-aspartate receptors in cholinergic interneurons. Acute in vitro application of human α-syn oligomers mimicked the synaptic effects observed ex vivo in PD models. CONCLUSIONS: We suggest that striatal cholinergic dysfunction, induced by a direct interaction between α-syn and GluN2D-expressing N-methyl-D-aspartate receptors, represents a precocious biological marker of the disease.

Cyclosporin promotes neurorestoration and cell replacement therapy in pre-clinical models of Parkinson's disease.

BACKGROUND: The early clinical trials using fetal ventral mesencephalic (VM) allografts in Parkinson's disease (PD) patients have shown efficacy (albeit not in all cases) and have paved the way for further development of cell replacement therapy strategies in PD. The preclinical work that led to these clinical trials used allografts of fetal VM tissue placed into 6-OHDA lesioned rats, while the patients received similar allografts under cover of immunosuppression in an α-synuclein disease state. Thus developing models that more faithfully replicate the clinical scenario would be a useful tool for the translation of such cell-based therapies to the clinic. RESULTS: Here, we show that while providing functional recovery, transplantation of fetal dopamine neurons into the AAV-α-synuclein rat model of PD resulted in smaller-sized grafts as compared to similar grafts placed into the 6-OHDA-lesioned striatum. Additionally, we found that cyclosporin treatment was able to promote the survival of the transplanted cells in this allografted state and surprisingly also provided therapeutic benefit in sham-operated animals. We demonstrated that delayed cyclosporin treatment afforded neurorestoration in three complementary models of PD including the Thy1-α-synuclein transgenic mouse, a novel AAV-α-synuclein mouse model, and the MPTP mouse model. We then explored the mechanisms for this benefit of cyclosporin and found it was mediated by both cell-autonomous mechanisms and non-cell autonomous mechanisms. CONCLUSION: This study provides compelling evidence in favor for the use of immunosuppression in all grafted PD patients receiving cell replacement therapy, regardless of the immunological mismatch between donor and host cells, and also suggests that cyclosporine treatment itself may act as a disease-modifying therapy in all PD patients.

Nurr1 and Retinoid X Receptor Ligands Stimulate Ret Signaling in Dopamine Neurons and Can Alleviate α-Synuclein Disrupted Gene Expression.

α-synuclein, a protein enriched in Lewy bodies and highly implicated in neurotoxicity in Parkinson's disease, is distributed both at nerve terminals and in the cell nucleus. Here we show that a nuclear derivative of α-synuclein induces more pronounced changes at the gene expression level in mouse primary dopamine (DA) neurons compared to a derivative that is excluded from the nucleus. Moreover, by RNA sequencing we analyzed the extent of genome-wide effects on gene expression resulting from expression of human α-synuclein in primary mouse DA neurons. The results implicated the transcription factor Nurr1 as a key dysregulated target of α-synuclein toxicity. Forced Nurr1 expression restored the expression of hundreds of dysregulated genes in primary DA neurons expressing α-synuclein, and therefore prompted us to test the possibility that Nurr1 can be pharmacologically targeted by bexarotene, a ligand for the retinoid X receptor that forms heterodimers with Nurr1. Although our data demonstrated that bexarotene was ineffective in neuroprotection in rats in vivo, the results revealed that bexarotene has the capacity to coregulate subsets of Nurr1 target genes including the receptor tyrosine kinase subunit Ret. Moreover, bexarotene was able to restore dysfunctional Ret-dependent neurotrophic signaling in α-synuclein-overexpressing mouse DA neurons. These data highlight the role of the Nurr1-Ret signaling pathway as a target of α-synuclein toxicity and suggest that retinoid X receptor ligands with appropriate pharmacological properties could have therapeutic potential in Parkinson's disease. SIGNIFICANCE STATEMENT: How α-synuclein, a protein enriched in Lewy bodies in Parkinson's disease, is causing neuropathology in dopamine neurons remains unclear. This study elucidated how α-synuclein is influencing gene expression and how Nurr1, a transcription factor known to protect dopamine neurons against α-synuclein toxicity, can counteract these effects. Moreover, given the protective role of Nurr1, this study also investigated how Nurr1 could be pharmacologically targeted via bexarotene, a ligand of Nurr1's heterodimerization partner retinoid X receptor (RXR). The results showed that RXR ligands could increase neurotrophic signaling, but provided a mixed picture of its potential in a Parkinson's disease rat model in vivo. However, this study clearly emphasized Nurr1's neuroprotective role and indicated that other RXR ligands could have therapeutic potential in Parkinson's disease.

NURR1 in Parkinson disease--from pathogenesis to therapeutic potential.

In Parkinson disease (PD), affected midbrain dopamine (DA) neurons lose specific dopaminergic properties before the neurons die. How the phenotype of DA neurons is normally established and the ways in which pathology affects the maintenance of cell identity are, therefore, important considerations. Orphan nuclear receptor NURR1 (NURR1, also known as NR4A2) is involved in the differentiation of midbrain DA neurons, but also has an important role in the adult brain. Emerging evidence indicates that impaired NURR1 function might contribute to the pathogenesis of PD: NURR1 and its transcriptional targets are downregulated in midbrain DA neurons that express high levels of the disease-causing protein α-synuclein. Clinical and experimental data indicate that disrupted NURR1 function contributes to induction of DA neuron dysfunction, which is seen in early stages of PD. The likely involvement of NURR1 in the development and progression of PD makes this protein a potentially interesting target for therapeutic intervention.

mTOR inhibition alleviates L-DOPA-induced dyskinesia in parkinsonian rats.

The development of dyskinesia upon chronic L-DOPA treatment is a major complication for the management of the motor symptoms in Parkinson's disease (PD) patients. Efforts are made to understand the underlying mechanisms and identify targets for the pharmacological alleviation of dyskinesia without affecting the therapeutic effect of L-DOPA. Previous studies have shown that the mTOR pathway is hyperactive in dyskinesia as a consequence of D1 receptor hypersensitivity. We investigated the effect of the FDA-approved mTOR inhibitor Temsirolimus (CCI-779), currently used in the clinic, on the development of LID and on the severity of already established LID in hemi-parkinsonian rats. Systemic delivery of CCI-779 prevented the development of LID and significantly alleviated the severity of dyskinesia in L-DOPA-primed animals. This was associated with a reduced activation of the mTOR pathway in striatal medium spiny neurons. Drugs with mTOR inhibiting activity that are actively developed in cancer research may be of interest for the management of LID in PD patients.

TFEB: Pathogenic role and therapeutic target in Parkinson disease.

Parkinson disease (PD) is characterized by the progressive loss of nigral dopamine neurons and the presence of accumulations containing the disease-causing protein SNCA/α-synuclein. Here we review our recent findings describing how SNCA impairs the function of the master regulator of the autophagy-lysosomal pathway (ALP), the transcription factor EB (TFEB), and that genetic or pharmacological stimulation of its activity promotes protection of dopamine neurons. These findings suggest that strategies aimed at enhancing autophagy-mediated degradation of SNCA may hold great promise for disease intervention in PD.

TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity.

The aggregation of α-synuclein plays a major role in Parkinson disease (PD) pathogenesis. Recent evidence suggests that defects in the autophagy-mediated clearance of α-synuclein contribute to the progressive loss of nigral dopamine neurons. Using an in vivo model of α-synuclein toxicity, we show that the PD-like neurodegenerative changes induced by excess cellular levels of α-synuclein in nigral dopamine neurons are closely linked to a progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of transcription factor EB (TFEB), a major transcriptional regulator of the autophagy-lysosome pathway. The changes in lysosomal function, observed in the rat model as well as in human PD midbrain, were reversed by overexpression of TFEB, which afforded robust neuroprotection via the clearance of α-synuclein oligomers, and were aggravated by microRNA-128-mediated repression of TFEB in both A9 and A10 dopamine neurons. Delayed activation of TFEB function through inhibition of mammalian target of rapamycin blocked α-synuclein induced neurodegeneration and further disease progression. The results provide a mechanistic link between α-synuclein toxicity and impaired TFEB function, and highlight TFEB as a key player in the induction of α-synuclein-induced toxicity and PD pathogenesis, thus identifying TFEB as a promising target for therapies aimed at neuroprotection and disease modification in PD.

Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons.

Developmental transcription factors important in early neuron specification and differentiation often remain expressed in the adult brain. However, how these transcription factors function to mantain appropriate neuronal identities in adult neurons and how transcription factor dysregulation may contribute to disease remain largely unknown. The transcription factor Nurr1 has been associated with Parkinson's disease and is essential for the development of ventral midbrain dopamine (DA) neurons. We used conditional Nurr1 gene-targeted mice in which Nurr1 is ablated selectively in mature DA neurons by treatment with tamoxifen. We show that Nurr1 ablation results in a progressive pathology associated with reduced striatal DA, impaired motor behaviors, and dystrophic axons and dendrites. We used laser-microdissected DA neurons for RNA extraction and next-generation mRNA sequencing to identify Nurr1-regulated genes. This analysis revealed that Nurr1 functions mainly in transcriptional activation to regulate a battery of genes expressed in DA neurons. Importantly, nuclear-encoded mitochondrial genes were identified as the major functional category of Nurr1-regulated target genes. These studies indicate that Nurr1 has a key function in sustaining high respiratory function in these cells, and that Nurr1 ablation in mice recapitulates early features of Parkinson's disease.

α-Synuclein-induced down-regulation of Nurr1 disrupts GDNF signaling in nigral dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) and its close relative neurturin are currently in clinical trials for neuroprotection in patients with Parkinson disease (PD). However, in animal models of PD, GDNF fails to protect nigral dopamine (DA) neurons against α-synuclein-induced neurodegeneration. Using viral vector delivery of human wild-type α-synuclein to nigral DA neurons in rats, we show that the intracellular response to GDNF is blocked in DA neurons that overexpress α-synuclein. This block is accompanied by reduced expression of the transcription factor Nurr1 and its downstream target, the GDNF receptor Ret. We found that Ret expression was also reduced in nigral DA neurons in PD patients. Conditional knockout of Nurr1 in mice resulted in reduced Ret expression and blockade of the response to GDNF, whereas overexpression of Nurr1 restored signaling, providing protection of nigral DA neurons against α-synuclein toxicity. These results suggest that Nurr1 is a regulator of neurotrophic factor signaling and a key player in the cellular defense against α-synuclein toxicity.

Impaired neurotransmission caused by overexpression of α-synuclein in nigral dopamine neurons.

We used in vivo amperometry to monitor changes in synaptic dopamine (DA) release in the striatum induced by overexpression of human wild-type α-synuclein in nigral DA neurons, induced by injection of an adeno-associated virus type 6 (AAV6)-α-synuclein vector unilaterally into the substantia nigra in adult rats. Impairments in DA release evolved in parallel with the development of degenerative changes in the nigrostriatal axons and terminals. The earliest change, seen 10 d after vector injection, was a marked, ≈50%, reduction in DA reuptake, consistent with an early dysfunction of the DA transporter that developed before any overt signs of axonal damage. At 3 wk, when the first signs of axonal damage were observed, the amount of DA released after a KCl pulse was reduced by 70-80%, and peak DA concentration was delayed, indicating an impaired release mechanism. At later time points, 8-16 wk, overall striatal innervation density was reduced by 60-80% and accompanied by abundant signs of axonal damage in the form of α-synuclein aggregates, axonal swellings, and dystrophic axonal profiles. At this stage DA release and reuptake were profoundly reduced, by 80-90%. The early changes in synaptic DA release induced by overexpression of human α-synuclein support the idea that early predegenerative changes in the handling of DA may initiate, and drive, a progressive degenerative process that hits the axons and terminals first. Synaptic dysfunction and axonopathy would thus be the hallmark of presymptomatic and early-stage Parkinson disease, followed by neuronal degeneration and cell loss, characteristic of more advanced stages of the disease.

Neuroprotection by neuropeptide Y in cell and animal models of Parkinson's disease.

This study was aimed to investigate the potential neuroprotective effect of neuropeptide Y (NPY) on the survival of dopaminergic cells in both in vitro and in animal models of Parkinson's disease (PD). NPY protected human SH-SY5Y dopaminergic neuroblastoma cells from 6-hydroxydopamine-induced toxicity. In rat and mice models of PD, striatal injection of NPY preserved the nigrostriatal dopamine pathway from degeneration as evidenced by quantification of (1) tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta, levels of (2) striatal tyrosine hydroxylase and dopamine transporter, (3) dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) as well as (4) rotational behavior. NPY had no neuroprotective effects in mice treated with Y(2) receptor antagonist or in transgenic mice deficient for Y(2) receptor suggesting that NPY effects are mediated through this receptor. Stimulation of Y(2) receptor by NPY triggered the activation of both the ERK1/2 and Akt pathways but did not modify levels of brain derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor. These results open new perspectives in neuroprotective therapies using NPY and suggest potential beneficial effects in PD.

GDNF fails to exert neuroprotection in a rat α-synuclein model of Parkinson's disease.

The neuroprotective effect of the glial cell line-derived neurotrophic factor has been extensively studied in various toxic models of Parkinson's disease. However, it remains unclear whether this neurotrophic factor can protect against the toxicity induced by the aggregation-prone protein α-synuclein. Targeted overexpression of human wild-type α-synuclein in the nigrostriatal system, using adeno-associated viral vectors, causes a progressive degeneration of the nigral dopamine neurons and the development of axonal pathology in the striatum. In the present study, we investigated, using different paradigms of delivery, whether glial cell line-derived neurotrophic factor can protect against the neurodegenerative changes and the cellular stress induced by α-synuclein. We found that viral vector-mediated delivery of glial cell line-derived neurotrophic factor into substantia nigra and/or striatum, administered 2-3 weeks before α-synuclein, was inefficient in preventing the wild-type α-synuclein-induced loss of dopamine neurons and terminals. In addition, glial cell line-derived neurotrophic factor overexpression did not ameliorate the behavioural deficit in this rat model of Parkinson's disease. Quantification of striatal α-synuclein-positive aggregates revealed that glial cell line-derived neurotrophic factor had no effect on α-synuclein aggregation. These data provide the evidence for the lack of neuroprotective effect of glial cell line-derived neurotrophic factor against the toxicity of human wild-type α-synuclein in an in vivo model of Parkinson's disease. The difference in neuroprotective efficacy of glial cell line-derived neurotrophic factor seen in our model and the commonly used neurotoxin models of Parkinson's disease, raises important issues pertinent to the interpretation of the results obtained in preclinical models of Parkinson's disease, and their relevance for the therapeutic use glial cell line-derived neurotrophic factor in patients with Parkinson's disease.

Environmental enrichment does not reduce the rewarding and neurotoxic effects of methamphetamine.

Abuse of amphetamine analogues, such as methamphetamine (METH), represents an important health problem because of their powerful addictive and neurotoxic effects. Abuse of METH induces dopamine neuron terminals loss and cell death in the striatum similar to what is found in other neurodegenerative processes. Exposing mice and rats to enriched environments (EE) has been shown to produce significant protective effects against drug-induced reward as well as against neurodegenerative processes. Here, we investigated whether exposure to EE could reduce the METH-induced reward and neurotoxicity. For this, we reared mice for 2 months during early stages of life in standard environments or EE and then, at adulthood, we tested the ability of METH to induce conditioned place preference and neurotoxicity. We found that, contrary to what we found with other drugs such as cocaine and heroin, EE was unable to reduce the rewarding effects of METH. In addition, contrary to what we found with other toxins such as MPTP, EE did not diminish the striatal neurotoxicity induced by METH (4 x 10 mg/kg) as measured by dopamine content, tyrosine hydroxylase protein levels and apoptosis. Our results demonstrate that the rewarding and neurotoxic effects of METH are not reduced by EE and highlight the great risks associated with the increased popularity of this drug amongst the young population.

Exogenous neuropeptide Y promotes in vivo hippocampal neurogenesis.

Adult neurogenesis mainly occurs in two brain regions, the subventricular zone and the dentate gyrus (DG) of the hippocampus. Neuropeptide Y (NPY) is widely expressed throughout the brain and is known to enhance in vitro hippocampal cell proliferation. Mice lacking either NPY or the Y1 receptor display lower levels of cell proliferation, thereby suggesting a role for NPY in basal in vivo neurogenesis. Here, we investigated whether exogenous NPY stimulates DG progenitors proliferation in vivo. We show that intracerebroventricular administration of NPY increases DG cell proliferation and promotes neuronal differentiation in C57BL/6 adult mice. In these mice, the proliferative effect of NPY is mediated by the Y1 and not the Y2 receptor, as a Y1 ([Leu(31) ,Pro(34) ]), but not a Y2 (NPY(3-36) ), receptor agonist enhanced proliferation. In addition, no NPY-induced DG cellular proliferation is observed following NPY injection when coadministered with a Y1 antagonist or in the Y1 receptor knockout mouse. These results are in line with data obtained in Y1(-/-) mice, demonstrating that NPY regulates in vivo hippocampal neurogenesis. © 2010 Wiley-Liss, Inc.

Viral vector-mediated overexpression of α-synuclein as a progressive model of Parkinson's disease.

The discovery of the role of α-synuclein in the pathogenesis of Parkinson's disease (PD) has opened new possibilities for the development of more authentic models of Parkinson's disease. Recombinant adeno-associated virus (AAV) and lentivirus (LV) vectors are efficient tools for expression of genes locally in subsets of neurons in the brain and can be used to express human wild-type or mutated α-synuclein selectively in midbrain dopamine neurons. Using this approach, it is possible to trigger extensive PD-like cellular and axonal pathologies in the nigrostriatal projection, involving abnormal protein aggregation, neuronal dysfunction, and cell death that develop progressively over time. Targeted overexpression of human α-synuclein in midbrain dopamine neurons, using AAV vectors, reproduces many of the characteristic features of the human disease and provides, for the first time, a model of progressive PD that can be applied to both rodents and primates.

Anatomical and functional reconstruction of the nigrostriatal pathway by intranigral transplants.

The main transplantation strategy in Parkinson's disease has been to place dopaminergic grafts not in their ontogenic site, the substantia nigra, but in their target area, the striatum with contrasting results. Here we have used green fluorescent protein transgenic mouse embryos as donors of ventral mesencephalic cells for transplantation into the pre-lesioned substantia nigra of an adult wild-type host. This allows distinguishing the transplanted cells and their projections from those of the host. Grafted cells integrated within the host mesencephalon and expressed the dopaminergic markers tyrosine hydroxylase, vesicular monoamine transporter 2 and dopamine transporter. Most of the dopaminergic cells within the transplant expressed the substantia nigra marker Girk2 while a lesser proportion expressed the ventral tegmental area marker calbindin. Mesencephalic transplants developed projections through the medial forebrain bundle to the striatum, increased striatal dopamine levels and restored normal behavior. Interestingly, only mesencephalic transplants were able to restore the nigrostriatal projections as dopamine neurons originating from embryonic olfactory bulb transplants send projections only in the close vicinity of the transplantation site that did not reach the striatum. Our results show for the first time the ability of intranigral foetal dopaminergic neurons grafts to restore the damaged nigrostriatal pathway in adult mice. Together with our previous findings of efficient embryonic transplantation within the pre-lesioned adult motor cortex, these results demonstrate that the adult brain is permissive to specific and long distance axonal growth. They further open new avenues in cell transplantation therapies applied for the treatment of neurodegenerative disorders such as Parkinson's disease.

Neuropeptide Y stimulates proliferation, migration and differentiation of neural precursors from the subventricular zone in adult mice.

The neuropeptide Y (NPY) is widely expressed in the central nervous system and has been shown to stimulate neurogenesis in the hippocampus and the olfactory epithelium. Here, we demonstrate that intracerebroventricular injection of NPY stimulates proliferation of neural precursors in the mice subventricular zone (SVZ), one the most neurogenic areas of the brain. Newly generated neuroblasts migrate through the rostral migratory stream to the olfactory bulb and also directly to the striatum, as evidenced by BrdU labelling and cell phenotyping. Using knock-out mice, specific NPY receptor agonists and antagonists, we report that this neuroproliferative effect is mediated by the Y1 receptor subtype that we found to be highly expressed in the SVZ both at the mRNA and protein levels. Our data suggest that stimulating endogenous SVZ neural stem cells by NPY may be of a potential interest in cell replacement based therapies of neurodegenerative diseases affecting the striatum such as Huntington's disease.