FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury.

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways.

Axonal Protection by Netarsudil, a ROCK Inhibitor, Is Linked to an AMPK-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration.

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve. Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated. Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration. Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.

The role of E3 ubiquitin ligases in synapse function in the healthy and diseased brain.

Ubiquitination is a key posttranslational modification for the controlled protein degradation and proteostasis. The substrate specificity is determined by a family of E3 ubiquitin ligases, which are encoded by more than 600 genes in the mammalian genome. Gain- or loss-of-function of a number of E3 genes results in neurodegeneration or neurodevelopmental disorders, affecting synapse function. This implies that the specific ubiquitination of synaptic substrates are of crucial importance for the normal neuronal network. In this review, we will summarize the history, current topics, and challenges in the field of ubiquitination-dependent regulations of synaptogenesis and synaptic transmission.

The Peptidyl-prolyl Isomerase Pin1 in Neuronal Signaling: from Neurodevelopment to Neurodegeneration.

The peptidyl-prolyl isomerase Pin1 is a unique enzyme catalyzing the isomerization of the peptide bond between phosphorylated serine-proline or threonine-proline motifs in proteins, thereby regulating a wide spectrum of protein functions, including folding, intracellular signaling, transcription, cell cycle progression, and apoptosis. Pin1 has been reported to act as a key molecular switch inducing cell-type-specific effects, critically depending on the different phosphorylation patterns of its targets within different biological contexts. While its implication in proliferating cells, and, in particular, in the field of cancer, has been widely characterized, less is known about Pin1 biological functions in terminally differentiated and post-mitotic neurons. Notably, Pin1 is widely expressed in the central and peripheral nervous system, where it regulates a variety of neuronal processes, including neuronal development, apoptosis, and synaptic activity. However, despite studies reporting the interaction of Pin1 with neuronal substrates or its involvement in specific signaling pathways, a more comprehensive understanding of its biological functions at neuronal level is still lacking. Besides its implication in physiological processes, a growing body of evidence suggests the crucial involvement of Pin1 in aging and age-related and neurodegenerative diseases, including Alzheimer's disease, Parkinson disease, frontotemporal dementias, Huntington disease, and amyotrophic lateral sclerosis, where it mediates profoundly different effects, ranging from neuroprotective to neurotoxic. Therefore, a more detailed understanding of Pin1 neuronal functions may provide relevant information on the consequences of Pin1 deregulation in age-related and neurodegenerative disorders.

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

Loss- or Gain-of-Function Mutations in ACOX1 Cause Axonal Loss via Different Mechanisms.

ACOX1 (acyl-CoA oxidase 1) encodes the first and rate-limiting enzyme of the very-long-chain fatty acid (VLCFA) ß-oxidation pathway in peroxisomes and leads to H2O2 production. Unexpectedly, Drosophila (d) ACOX1 is mostly expressed and required in glia, and loss of ACOX1 leads to developmental delay, pupal death, reduced lifespan, impaired synaptic transmission, and glial and axonal loss. Patients who carry a previously unidentified, de novo, dominant variant in ACOX1 (p.N237S) also exhibit glial loss. However, this mutation causes increased levels of ACOX1 protein and function resulting in elevated levels of reactive oxygen species in glia in flies and murine Schwann cells. ACOX1 (p.N237S) patients exhibit a severe loss of Schwann cells and neurons. However, treatment of flies and primary Schwann cells with an antioxidant suppressed the p.N237S-induced neurodegeneration. In summary, both loss and gain of ACOX1 lead to glial and neuronal loss, but different mechanisms are at play and require different treatments.

CD38 in Neurodegeneration and Neuroinflammation.

Neurodegenerative diseases are characterized by neuronal degeneration as well as neuroinflammation. While CD38 is strongly expressed in brain cells including neurons, astrocytes as well as microglial cells, the role played by CD38 in neurodegeneration and neuroinflammation remains elusive. Yet, CD38 expression increases as a consequence of aging which is otherwise the primary risk associated with neurodegenerative diseases, and several experimental data demonstrated that CD38 knockout mice are protected from neurodegenerative and neuroinflammatory insults. Moreover, nicotinamide adenine dinucleotide, whose levels are tightly controlled by CD38, is a recognized and potent neuroprotective agent, and NAD supplementation was found to be beneficial against neurodegenerative diseases. The aims of this review are to summarize the physiological role played by CD38 in the brain, present the arguments indicating the involvement of CD38 in neurodegeneration and neuroinflammation, and to discuss these observations in light of CD38 complex biology.

Central aromatization: A dramatic and responsive defense against threat and trauma to the vertebrate brain.

Aromatase is the requisite and limiting enzyme in the production of estrogens from androgens. Estrogens synthesized centrally have more recently emerged as potent neuroprotectants in the vertebrate brain. Studies in rodents and songbirds have identified key mechanisms that underlie both; the injury-dependent induction of central aromatization, and the protective effects of centrally synthesized estrogens. Injury-induced aromatase expression in astrocytes occurs following a broad range of traumatic brain damage including excitotoxic, penetrating, and concussive injury. Responses to neural insult such as edema and inflammation involve signaling pathways the components of which are excellent candidates as inducers of this astrocytic response. Finally, estradiol from astrocytes exerts a paracrine neuroprotective influence via the potent inhibition of inflammatory pathways. Taken together, these data suggest a novel role for neural aromatization as a protective mechanism against the threat of inflammation and suggests that central estrogen provision is a wide-ranging neuroprotectant in the vertebrate brain.

Disrupted glycosylation of lipids and proteins is a cause of neurodegeneration.

Glycosyltransferases represent a large family of enzymes that catalyse the biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates. A number of studies have implicated glycosyltransferases in the pathogenesis of neurodegenerative diseases but differentiating cause from effect has been difficult. We have recently discovered that mutations proximal to the substrate binding site of glycosyltransferase 8 domain containing 1 (GLT8D1) are associated with familial amyotrophic lateral sclerosis (ALS). We demonstrated that ALS-associated mutations reduce activity of the enzyme suggesting a loss-of-function mechanism that is an attractive therapeutic target. Our work is the first evidence that isolated dysfunction of a glycosyltransferase is sufficient to cause a neurodegenerative disease, but connection between neurodegeneration and genetic variation within glycosyltransferases is not new. Previous studies have identified associations between mutations in UGT8 and sporadic ALS, and between ST6GAL1 mutations and conversion of mild cognitive impairment into clinical Alzheimer's disease. In this review we consider potential mechanisms connecting glycosyltransferase dysfunction to neurodegeneration. The most prominent candidates are ganglioside synthesis and impaired addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) groups to proteins important for axonal and synaptic function. Special consideration is given to examples where genetic mutations within glycosyltransferases are associated with neurodegeneration in recognition of the fact that these changes are likely to be upstream causes present from birth.

Neuronally Enriched RUFY3 Is Required for Caspase-Mediated Axon Degeneration.

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.

Low-Level Primary Blast Induces Neuroinflammation and Neurodegeneration in Rats.

OBJECTIVE: Mild blast traumatic brain injury is commonly prevalent in modern combat casualty care and has been associated with the development of neurodegenerative conditions. However, whether primary lower level blast overpressure (LBOP) causes neurodegeneration and neuroinflammation remains largely unknown. The aim of our present study was to determine whether LBOP can cause neuroinflammation and neurodegeneration. METHODS: Anesthetized rats were randomly assigned to LBOP group (70 kPa, n = 5) or sham group (without blast, n = 5). Histopathological and cytokine changes in brain tissue at 5 days post-injury were evaluated by hematoxylin-eosin staining and Bioplex assay, respectively. RESULTS: Histopathological assessment revealed neuronal degeneration and increased density of inflammatory cells in frontal and parietal cortex, hippocampus and thalamus in rats exposed to LBOP. LBOP exposure significantly elevated levels of pro-inflammatory cytokines (EPO, IL-1ß, IL-6, IL-12, IL-18, and TNF-α) and chemokines (GRO and RANTES) as well as of an anti-inflammatory cytokine (IL-13) in the frontal cortex. CONCLUSIONS: This study reveals a role of neuroinflammation in neurodegeneration after mild blast traumatic brain injury. Therapies that target this process might in warfighters might function either by attenuating the development of post-traumatic stress disorder, chronic traumatic encephalopathy and Alzheimer's disease, or by slowing their progression.

NADPH oxidase regulates paraquat and maneb-induced dopaminergic neurodegeneration through ferroptosis.

The activation of NADPH oxidase contributes to dopaminergic neurodegeneration induced by paraquat and maneb, two concurrently used pesticides in agriculture. However, the mechanisms remain unclear. Ferroptosis, a recently recognized form of regulated cell death, has been implicated in the pathogenesis of multiple neurodegenerative diseases. This study is designed to investigate whether ferroptosis is involved in NADPH oxidase-regulated dopaminergic neurotoxicity. In vitro study showed that paraquat and maneb exposure induced ferroptosis in SHSY5Y dopaminergic cells, which was associated with activation of NADPH oxidase. Inhibition of NADPH oxidase by apocynin or diphenyleneiodonium (DPI), two widely used NADPH oxidase inhibitors mitigated paraquat and maneb-induced ferroptotic cell death. Consistently, stimulating activation of NADPH oxidase by phorbol myristate acetate (PMA) or supplementation of H2O2 exacerbated ferroptosis in paraquat and maneb-treated SHSY5Y cells. Mechanistic inquiry revealed that NADPH oxidase activation elicited lipid peroxidation, a main driving force for ferroptosis, since both apocynin and DPI greatly reduced MDA contents and simultaneously recovered levels of glutathione and glutathione peroxidase 4 (GPX4) in paraquat and maneb-treated SHSY5Y cells. The contribution of NADPH oxidase on ferroptosis of dopaminergic neurons was further verified in vivo by showing reduced iron content, lipid peroxidation, neuroinflammation and dopaminergic neurodegeneration, which are all involved in ferroptosis, in combined apocynin and paraquat and maneb-treated mice compared with paraquat and maneb alone group. Altogether, our findings showed that NADPH oxidase contributed to paraquat and maneb-induced dopaminergic neurodegeneration through ferroptosis, providing a novel mechanism for pesticide-induced dopaminergic neurotoxicity.

MAP4K4 Activation Mediates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons (MNs). To date, its underlying mechanisms have yet to be clarified completely, and there are no truly effective treatments. Here, we show that MAP4K4, a MAP kinase family member, regulates MN death, with its suppression not only promoting survival but preventing neurite degeneration and decreasing mutant SOD1 levels through autophagy activation. Moreover, we report that MAP4K4 signaling specifically modulates MN viability via phosphorylated JNK3 and activation of the canonical c-Jun apoptotic pathway. Finally, we show the feasibility of MAP4K4 as a drug target by using an available MAP4K4-specific inhibitor, which improves survival of ESC and/or iPSC-derived MNs and MNs cultured from mouse spinal cords. In summary, our studies highlight a MAP4K4-initiated signaling cascade that induces MN degeneration, shedding light on the mechanism underlying MN degeneration and providing a druggable target for ALS therapeutics.

Neurodegeneration in SCA14 is associated with increased PKCγ kinase activity, mislocalization and aggregation.

Spinocerebellar ataxia type 14 (SCA14) is a subtype of the autosomal dominant cerebellar ataxias that is characterized by slowly progressive cerebellar dysfunction and neurodegeneration. SCA14 is caused by mutations in the PRKCG gene, encoding protein kinase C gamma (PKCγ). Despite the identification of 40 distinct disease-causing mutations in PRKCG, the pathological mechanisms underlying SCA14 remain poorly understood. Here we report the molecular neuropathology of SCA14 in post-mortem cerebellum and in human patient-derived induced pluripotent stem cells (iPSCs) carrying two distinct SCA14 mutations in the C1 domain of PKCγ, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKCγ in both patient iPSCs and cerebellum. PKCγ aggregates were not efficiently targeted for degradation. Moreover, mutant PKCγ was found to be hyper-activated, resulting in increased substrate phosphorylation. Together, our findings demonstrate that a combination of both, loss-of-function and gain-of-function mechanisms are likely to underlie the pathogenesis of SCA14, caused by mutations in the C1 domain of PKCγ. Importantly, SCA14 patient iPSCs were found to accurately recapitulate pathological features observed in post-mortem SCA14 cerebellum, underscoring their potential as relevant disease models and their promise as future drug discovery tools.

An axonal stress response pathway: degenerative and regenerative signaling by DLK.

Signaling through the dual leucine zipper-bearing kinase (DLK) is required for injured neurons to initiate new axonal growth; however, activation of this kinase also leads to neuronal degeneration and death in multiple models of injury and neurodegenerative diseases. This has spurred current consideration of DLK as a candidate therapeutic target, and raises a vital question: in what context is DLK a friend or foe to neurons? Here, we review our current understanding of DLK's function and mechanisms in regulating both regenerative and degenerative responses to axonal damage and stress in the nervous system.

Jnk2 deficiency increases the rate of glaucomatous neurodegeneration in ocular hypertensive DBA/2J mice.

The cJun N-terminal kinases (JNKs; JNK1, JNK2, and JNK3) promote degenerative processes after neuronal injury and in disease. JNK2 and JNK3 have been shown to promote retinal ganglion cell (RGC) death after optic nerve injury. In their absence, long-term survival of RGC somas is significantly increased after mechanical optic nerve injury. In glaucoma, because optic nerve damage is thought to be a major cause of RGC death, JNKs are an important potential target for therapeutic intervention. To assess the role of JNK2 and JNK3 in an ocular hypertensive model of glaucoma, null alleles of Jnk2 and Jnk3 were backcrossed into the DBA/2J (D2) mouse. JNK activation occurred in RGCs following increased intraocular pressure in D2 mice. However, deficiency of both Jnk2 and Jnk3 together did not lessen optic nerve damage or RGC death. These results differentiate the molecular pathways controlling cell death in ocular hypertensive glaucoma compared with mechanical optic nerve injury. It is further shown that JUN, a pro-death component of the JNK pathway in RGCs, can be activated in glaucoma in the absence of JNK2 and JNK3. This implicates JNK1 in glaucomatous RGC death. Unexpectedly, at younger ages, Jnk2-deficient mice were more likely to develop features of glaucomatous neurodegeneration than D2 mice expressing Jnk2. This appears to be due to a neuroprotective effect of JNK2 and not due to a change in intraocular pressure. The Jnk2-deficient context also unmasked a lesser role for Jnk3 in glaucoma. Jnk2 and Jnk3 double knockout mice had a modestly increased risk of neurodegeneration compared with mice only deficient in Jnk2. Overall, these findings are consistent with pleiotropic effects of JNK isoforms in glaucoma and suggest caution is warranted when using JNK inhibitors to treat chronic neurodegenerative conditions.

The deletion of dicer in mature myelinating glial cells causes progressive axonal degeneration but not overt demyelination in adult mice.

Myelinating glial cells (MGCs), oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS), generate myelin sheaths that insulate axons. After myelination is completed in adulthood, MGC functions independent from myelin are required to support axon survival, but the underlying mechanisms are still unclear. Dicer is a key enzyme that is responsible for generating functional micro-RNAs (miRNAs). Despite the importance of Dicer in initiating myelination, the role of Dicer in mature MGCs is still unclear. Here, Dicer was specifically deleted in mature MGCs in 2-month old mice (PLP-CreERT; Dicer fl/fl) by tamoxifen administration. Progressive motor dysfunction was observed in the Dicer conditional knockout mice, which displayed hind limb ataxia at 3 months post recombination that deteriorated into paralysis within 5 months. Massive axonal degeneration/atrophy in peripheral nerves was responsible for this phenomenon, but overt demyelination was not observed in either the CNS or PNS. In contrast to the PNS, signs of axonal degeneration were not observed in the CNS of these animals. We induced a Dicer deletion in oligodendroglia at postnatal day 5 in NG2-CreERT; Dicer fl/fl mice to evaluate whether Dicer expression in OLs is essential for axonal survival. Dicer deletion in oligodendroglia did not cause motor dysfunction at the age of 7 months. Neither axonal atrophy nor demyelination was observed in the CNS. Based on our results, Dicer expression in SCs is required to maintain axon integrity in adult PNS, and Dicer is dispensable for maintaining myelin sheaths in MGCs.

Myeloperoxidase Associates With Degenerative Remodeling and Rupture of the Saccular Intracranial Aneurysm Wall.

Rupture of a saccular intracranial aneurysm (sIA) is often fatal. Thus, early detection of rupture-prone sIAs is vital. Myeloperoxidase (MPO), derived mainly from neutrophils, associates with sIA rupture, and therefore its role in sIA pathogenesis warrants further studies. We analyzed MPO and its association with other histological markers in 36 (16 unruptured and 20 ruptured) sIA samples by immunohistochemistry. MPO was present in all studied sIAs, and its expression associated with wall inflammatory cell infiltrations (r = 0.50, 0.63, and 0.75, all p ≤ 0.002), degenerative remodeling (p = 0.002) and rupture (p = 0.003). MPO associated strongly with the presence of organized luminal thrombi (p < 0.001), which also stained positive for MPO. Polymorphonuclear MPO+ cells were detected in the sIA walls, indicating neutrophils as MPO-source. MPO correlated strongly with accumulation of oxidized lipids (r = 0.67, p < 0.001) and loss of smooth muscle cells (r = -0.68, p < 0.001), suggesting that MPO is a relevant source of oxidative stress leading to cell death in the sIA wall. Furthermore, MPO associated with erythrocyte fragmentation (r = 0.74, p < 0.001) and iron deposition (p = 0.041), 2 outcomes known to amplify MPO-dependent oxidative stress. Taken together, these results suggest that MPO associates with degenerative remodeling predisposing to sIA wall rupture and may serve as a biomarker of a rupture-prone sIA wall.

Biological function of Lemur tyrosine kinase 2 (LMTK2): implications in neurodegeneration.

Neurodegenerative disorders are frequent, incurable diseases characterised by abnormal protein accumulation and progressive neuronal loss. Despite their growing prevalence, the underlying pathomechanism remains unclear. Lemur tyrosine kinase 2 (LMTK2) is a member of a transmembrane serine/threonine-protein kinase family. Although it was described more than a decade ago, our knowledge on LMTK2's biological functions is still insufficient. Recent evidence has suggested that LMTK2 is implicated in neurodegeneration. After reviewing the literature, we identified three LMTK2-mediated mechanisms which may contribute to neurodegenerative processes: disrupted axonal transport, tau hyperphosphorylation and enhanced apoptosis. Moreover, LMTK2 gene expression is decreased in an Alzheimer's disease mouse model. According to these features, LMTK2 might be a promising therapeutic target in near future. However, further investigations are required to clarify the exact biological functions of this unique protein.

The Lipase Activity of Phospholipase D2 is Responsible for Nigral Neurodegeneration in a Rat Model of Parkinson's Disease.

Phospholipase D2 (PLD2), an enzyme involved in vesicle trafficking and membrane signaling, interacts with α-synuclein, a protein known to contribute in the development of Parkinson disease (PD). We previously reported that PLD2 overexpression in rat substantia nigra pars compacta (SNc) causes a rapid neurodegeneration of dopamine neurons, and that α-synuclein suppresses PLD2-induced nigral degeneration (Gorbatyuk et al., 2010). Here, we report that PLD2 toxicity is due to its lipase activity. Overexpression of a catalytically inactive mutant (K758R) of PLD2 prevents the loss of dopaminergic neurons in the SNc and does not show signs of toxicity after 10 weeks of overexpression. Further, mutant K758R does not affect dopamine levels in the striatum. In contrast, mutants that prevent PLD2 interaction with dynamin or growth factor receptor bound protein 2 (Grb2) but retained lipase activity, continued to show rapid neurodegeneration. These findings suggest that neither the interaction of PLD2 with dynamin, which has a role in vesicle trafficking, nor the PLD2 interaction with Grb2, which has multiple roles in cell cycle control, chemotaxis and activation of tyrosine kinase complexes, are the primary cause of neurodegeneration. Instead, the synthesis of phosphatidic acid (the product of PLD2), which is a second messenger in multiple cellular pathways, appears to be the key to PLD2 induced neurodegeneration. The fact that α-synuclein is a regulator of PLD2 activity suggests that regulation of PLD2 activity could be important in the progression of PD.