Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway.

Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

Identification through high-throughput screening of 4'-methoxyflavone and 3',4'-dimethoxyflavone as novel neuroprotective inhibitors of parthanatos.

BACKGROUND AND PURPOSE: The current lack of disease-modifying therapeutics to manage neurological and neurodegenerative conditions justifies the development of more efficacious agents. One distinct pathway leading to neuronal death in these conditions and which represents a very promising and attractive therapeutic target is parthanatos, involving overactivation of PARP-1. We therefore sought to identify small molecules that could be neuroprotective by targeting the pathway. EXPERIMENTAL APPROACH: Using HeLa cells, we developed and optimized an assay for high-throughput screening of about 5120 small molecules. Structure-activity relationship (SAR) study was carried out in HeLa and SH-SY5Y cells for molecules related to the initial active compound. The neuroprotective ability of each active compound was tested in cortical neuronal cultures. KEY RESULTS: 4'-Methoxyflavone (4MF) showed activity by preventing the decrease in cell viability of HeLa and SH-SY5Y cells caused by the DNA-alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which induces parthanatos. A similar compound from the SAR study, 3',4'-dimethoxyflavone (DMF), also showed significant activity. Both compounds reduced the synthesis and accumulation of poly (ADP-ribose) polymer and protected cortical neurones against cell death induced by NMDA. CONCLUSIONS AND IMPLICATIONS: Our data reveal additional neuroprotective members of the flavone class of flavonoids and show that methoxylation of the parent flavone structure at position 4' confers parthanatos-inhibiting activity while additional methoxylation at position 3', reported by others to improve metabolic stability, does not destroy the activity. These molecules may therefore serve as leads for the development of novel neurotherapeutics for the management of neurological and neurodegenerative conditions.

Myoclonus-dystonia and Silver-Russell syndrome resulting from maternal uniparental disomy of chromosome 7.

Myoclonus-dystonia (M-D) is a movement disorder that is often associated with mutations in epsilon-sarcoglycan (SGCE), a maternally imprinted gene at 7q21.3. We report a 24-year-old male with short stature (<5th percentile) and a movement disorder clinically consistent with M-D. Single nucleotide polymorphism (SNP) array did not identify significant copy number changes, but revealed three long continuous stretches of homozygosity on chromosome 7 suggestive of uniparental disomy. Parental SNP arrays confirmed that the proband had maternal uniparental disomy of chromosome 7 (mUPD7) with regions of heterodisomy and isodisomy. mUPD7 is the cause of approximately 5-10% of Silver-Russell syndrome (SRS), a disorder characterized by prenatal and postnatal growth retardation. Although SRS was not suspected in our patient, these findings explain his short stature. SGCE methylation testing showed loss of the unmethylated paternal allele. Our findings provide a unifying diagnosis for his short stature and M-D and help to optimize his medication regimen. In conclusion, we show that M-D is a clinical feature that may be associated with SRS due to mUPD7. Individuals with mUPD7 should be monitored for the development of movement disorders. Conversely, individuals with M-D and short stature should be evaluated for SRS.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

Spatial and functional relationship between poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in the brain.

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus.

Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.

Mining for survival genes.

Many stressful, but not lethal, stimuli activate endogenous protective mechanisms that significantly decrease the degree of injury to subsequent injurious stimuli. This protective mechanism is termed preconditioning and tolerance. It occurs across organ systems including the brain and nervous system. Preconditioning has been investigated in cell and animal models and recently been shown to potentially occur in human brain. Learning more about these powerful endogenous neuroprotective mechanisms could help identify new approaches to treat patients with stroke and other central nervous system disorders or injury. Cell and animal models are helping us to better understand the network response of gene and protein expression that activates the neuroprotective response.

Parkin and defective ubiquitination in Parkinson's disease.

Parkinson's Disease (PD) is a common neurodegenerative disorder that is characterized by the progressive loss of dopamine (DA) neurons. Accompanying the loss the of DA neurons is the accumulation of Lewy bodies and neurites, intracytoplasmic proteinaceous inclusions that contain alpha-synuclein, synphilin-1, components of the ubiquitin proteasomal pathway and parkin. Recent advances indicate that PD is due in some individuals to genetic mutations in alpha-synuclein, DJ-1, PINK-1, LRRK2, and parkin. Understanding the molecular mechanisms by which mutations in familial-linked genes cause PD holds great promise for unraveling the mechanisms by which DA neurons degenerate in PD. Parkin is E3-ubiquitin-protein ligase that ubiquitinates itself and promotes its own degradation. Familial associated mutations of parkin have impaired ubiquitin ligase function suggesting that this may be the cause of familial autosomal recessive PD. Parkin might be required for formation of Lewy bodies as Lewy bodies are absent in patients with parkin mutations. Parkin interacts with and ubiquitinates the alpha-synuclein interacting protein, synphilin-1. Formation of Lewy-body-like ubiquitin-positive cytosolic inclusions occurs upon coexpression of alpha-synuclein, synphilin-1 and parkin. Nitric oxide inhibits Parkin's E-3 ligase activity and its protective function by nitric oxide through S-nitrosylation both in vitro and in vivo. Nitrosative and oxidative stress link parkin function with the more common sporadic form of Parkinson's disease and the related alpha-synucleinopathy, DLBD. Development of new therapies for PD and other disorders associated with nitrosative and oxidative stress may follow the elucidation of the pathways by which NO S-nitrosylates and inhibits parkin. Moreover, parkin and alpha-synuclein are linked in common pathogenic mechanism through their interaction with synphilin-1 and parkin may be important for the formation of Lewy bodies.

EndoG is dispensable in embryogenesis and apoptosis.

The mitochondrial protein, endonuclease G (EndoG), is one of the endonucleases implicated in DNA fragmentation during apoptosis. It has been shown to translocate from the mitochondria to the nucleus upon cell death stimuli. These observations suggest that EndoG is a mitochondrial cell death effector, and that it possibly acts as a cell death nuclease, similar to DNA fragmentation factor. To better understand the role of EndoG in development and apoptosis, we generated EndoG null mice by homologous gene targeting without disruption of D2Wsu81e. EndoG null mice are viable and develop to adulthood with no obvious abnormalities. Fibroblasts generated from the EndoG null mice show no difference in susceptibility when induced to die by a variety of intrinsic and extrinsic apoptotic stimuli. Additionally, EndoG null mice are equally sensitive to excitotoxic stress. These data suggest that EndoG is not essential for early embryogenesis and apoptosis.

Nitric oxide, S-nitrosylation and neurodegeneration.

Nitric oxide is a critically important signaling molecule, controlling a wide range of pathways and biological processes. Highly reactive nitric oxide mediates its function through reaction with different molecules directly or indirectly. One of these modifications is the S-nitrosylation of cysteine residues in proteins. S-nitrosylation is emerging as an important redox signaling mechanism and has been found to regulate a broad range of biologic, physiologic and cellular functions. One of the major findings in this area recently is the linkage of nitrosative stress to various neurodegenerative disorders. Oxidative stress has long been regarded as a prime mediator in the development of neurodegeneration as various indices of oxidative stress are readily observed in postmortem studies. A causative role for nitrosative stress in neurodegeneration is just now being appreciated. The direct connection of S-nitrosylation to the pathogenesis of Parkinson's disease in recent studies further provide insights into how imbalance in nitric oxide metabolism can contribute to the development of selective injury and disease.

Poly(ADP-ribosyl)ation regulation of life and death in the nervous system.

Poly(ADP-ribosyl)ation is required by multicellular eukaryotes to ensure genomic integrity under conditions of mild to moderate genotoxic stress. However, severe stress following acute neuronal injury causes overactivation of poly(ADP-ribose) polymerase-1, which results in unregulated poly(ADP-ribose) (PAR) synthesis and widespread neuronal cell death. Once thought to be a necrotic cell death resulting from energy failure, PARP-1 activation is now known to induce the nuclear translocation of apoptosis-inducing factor, which results in caspase-independent cell death. Conversely, poly(ADP-ribose) glycohydrolase, once thought to contribute to neuronal injury, now appears to have a protective role as demonstrated by recent studies utilizing gene disruption technology. Thus, the emerging mechanism dictating the fate of neurons appears to involve the regulation of PAR levels in neurons. Therefore, therapies targeting poly(ADP-ribosyl)ation in the treatment of neurodegenerative conditions such as stroke and Parkinson's disease are required to inhibit PAR synthesis and/or facilitate its degradation.

Cortical interneurons become activated by deafferentation and instruct the apoptosis of pyramidal neurons.

Unlike peripheral nervous system neurons and certain groups of nerve cells in the CNS, cortical projection neurons are tolerant of axonal lesions. This resistance is incongruent with the massive death of pyramidal neurons in age-associated neurodegenerative diseases that proceed along corticocortical connections. Some insights have emerged from our previous work showing that pyramidal cells in piriform cortex undergo classical apoptosis within 24 h after bulbectomy via transsynaptic, but not retrograde, signaling. These findings allow the investigation of cellular and molecular changes that take place in the context of experimental cortical degeneration. In the present study, we show that the transsynaptic death of pyramidal neurons in piriform cortex is a nitric oxide-mediated event signaled by activated interneurons in layer I. Thus, we demonstrate that cortical interneurons play an essential role in transducing injury to apoptotic signaling that selectively targets pyramidal neurons. We propose that this mechanism may be generic to cortical degenerations and amenable to therapeutic interventions.

Psychiatric comorbidities in patients with Parkinson disease and psychosis.

OBJECTIVE: To determine the prevalence and impact of comorbid psychiatric disturbances in Parkinson disease (PD) patients with psychosis. METHODS: Subject data were derived from a research database of 116 PD patients participating in standardized motor, cognitive, psychiatric, and caregiver assessments. RESULTS: There were 25 patients (22%) with psychosis manifest as hallucinations (n = 9), delusions (n = 1), or hallucinations and delusions (n = 15) and 25 patients (22%) who had no current or past psychiatric comorbidities (PDN). In the psychotic group, 44% had psychosis only (PSY), and 56% had psychosis plus at least one other comorbid psychiatric disturbance (PSY+), including depressive disorders (71%), anxiety disorders (21%), apathetic syndromes (14%), and delirium (14%). There were no differences in age, sex, education, or age onset or duration of PD among the PSY, PSY+, and PDN groups. Both psychotic groups had greater motor, functional, and frontal cognitive deficits and increased caregiver burden scores relative to PDN. PSY+ showed greater global and selective cognitive deficits compared to PDN. Psychosis was a primary predictor of caregiver burden, whereas depressive symptoms indirectly enhanced motor impairments. CONCLUSIONS: Nonpsychotic psychiatric disturbances, especially affective disturbances, are common comorbidities in PD patients with psychosis and warrant clinical attention to reduce morbidity and caregiver distress.

SEPT5_v2 is a parkin-binding protein.

Mutations in parkin are associated with various inherited forms of Parkinson's disease (PD). Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation. The substrates of parkin-mediated ubiquitination have yet to be completely identified. Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein. SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination. SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains. Several lines of evidence have validated the putative link between parkin and SEPT5_v2. Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates. Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins. These findings suggest that an important relationship exists between parkin and septins.

Neuroprotective agents for clinical trials in Parkinson's disease: a systematic assessment.

BACKGROUND: Current therapies for PD ameliorate symptoms in the early phases of disease but become less effective over time, as the underlying disease progresses. Therapies that slow the progression of PD are needed. However, there have been relatively few clinical trials aimed at demonstrating neuroprotection. The authors sought to identify potential neuroprotective agents for testing in clinical trials. METHODS: First a broad array of compounds were identified by working with clinicians and researchers in academics and industry. Specific criteria were drafted for drug evaluation, including scientific rationale, blood-brain barrier penetration, safety and tolerability, and evidence of efficacy in animal models or humans. Agents were prioritized based on these criteria. RESULTS: The authors identified 59 potential neuroprotective compounds, proposed by 42 clinicians and scientists from 13 countries. After systematic reviews using the specified criteria they found 12 compounds to be attractive candidates for further clinical trials in PD. CONCLUSIONS: Several potential neuroprotective compounds, representing a wide range of mechanisms, are available and merit further investigation in PD.

Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease.

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.

Neuroimmunophilins: novel neuroprotective and neuroregenerative targets.

Cyclosporin A (CsA) and FK506 (tacrolimus) are immunosuppresants that are widely used in organ transplantation. CsA is an 11-member cyclic peptide, whereas FK506 is a macrolide antibiotic. Recently, these powerful and useful compounds have become of great interest to neuroscientists for their unique neuroprotective and neuroregenerative effects. These drugs and nonimmunosuppressive analogs protect neurons from the effects of glutamate excitotoxicity, focal ischemia, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic cell death. They also stimulate functional recovery of neurons in a variety of neurologic injury paradigms. These drugs exert their effects via immunophilins, the protein receptors for these agents. The immunophilin ligands show particular promise as a novel class of neuroprotective and neuroregenerative agents that have the potential to treat a variety of neurologic disorders.

Identification by mRNA differential display of two up-regulated genes as candidate mediators of AIDS dementia.

BACKGROUND: In the dementia associated with acquired immunodeficiency syndrome (AIDS), indirect pathomechanisms are important mediators of progressive neuronal injury and variable candidate molecules of potential pathogenetic importance have been identified. MATERIALS AND METHODS: In an attempt to characterize additional mediators of human immunodeficiency virus type 1 (HIV-1)-induced neurotoxicity in vivo we have adapted the mRNA differential display technique to monitor the gene expression pattern in postmortem cortical tissue from AIDS patients with (n = 7) and without (n = 8) cognitive impairment as well as from HIV-1 seronegative controls (n = 4). RESULTS: Out of 29 differentially expressed cDNAs, two cDNA clones had confirmed variation of transcriptional regulation as assessed by reverse Northern analysis and gene-specific reverse transcription polymerase chain reaction (RT-PCR) and were up-regulated in the cortex of patients with AIDS dementia. Nucleotide sequence analysis of the two cDNAs identified known genes not previously associated with the pathogenesis of AIDS dementia, including the neurotrophin receptor tyrosine kinase receptor B (TrkB) and the potassium channel human open rectifyer K+ channel (ORK) homologous open reading frame (HOHO1). CONCLUSIONS: The altered expression of these transcripts may contribute to AIDS dementia through the enhancement of microglial activation and immunologic nitric oxide synthase (iNOS) activity by abnormal neurotrophic regulation and interference with membrane excitability through disturbance of local ion homeostasis.

Neuroimmunophilin ligands exert neuroregeneration and neuroprotection in midbrain dopaminergic neurons.

Immunosuppressant drugs, like FK506, and nonimmunosuppressant compounds like, GPI1046 and L685818, are immunophilin ligands that specifically bind to immunophilins, like FK506 binding protein 12 (FKBP12). Several lines of evidence show that these ligands exert neurotrophic properties in neural injury models and in PC12 cells. However, the mechanism of the neurotrophic function of the immunophilin ligands is poorly known. In the present study, we use MPP+ and 6-OHDA toxicity models to examine both neuroprotective and neuroregenerative effects of immunophilin ligands on primary cultures of midbrain dopaminergic neurons. We find that FK506, GPI1046 and L685818 at concentrations from 0.01 to 1 microM partially, but significantly, protect dopaminergic neurons against both MPP+ and 6-OHDA toxicity. By Western blot analysis, we also find that all three compounds prevent tyrosine hydroxylase (TH) loss induced by MPP+ and 6-OHDA treatments. Morphologic analysis of dopaminergic neurons, by immunocytochemistry, shows that MPP+ and 6-OHDA cause the retraction and loss of neuronal processes, while FK506, GPI1046 and L685818 promote regeneration of these processes as indicated by increases in process number and length. To examine if FKBP12 is required for neurotrophic effects of immunophilin ligands, we cultured dopaminergic neurons from FKBP12 knockout mice and find that FK506 still protects dopaminergic neurons against MPP+ toxicity. These results suggest that FKBP12 is not essential for the neurotrophic properties of immunophilin ligands, and immunophilin ligands are a new class of neuroprotective and neuroregenerative agents that may have therapeutic potential in a variety of neurological disorders.