Genetic analysis of dopaminergic neuron survival

Pathological changes in the dopaminergic system account for a number of devastating illnesses including schizophrenia, psychosis, depression, addiction, obsessive compulsive disorder or the most well known Parkinson’s disease (PD). The nigrostriatal pathway is an important component of the dopaminergic (DA) system mediating voluntary movement and originates in the ventral midbrain from where substantia nigra pars compacta (SN) neurons send their axons to the dorsal striatum. Massive loss of SN neurons as seen in PD leads to postural imbalance, rigidity, tremor and bradykinesia, however, the precise mechanisms involved in the maintenance and the demise of SN neurons are poorly understood. Endogenous neurotrophic factors such as the Glial cell line-derived neurotrophic factor (GDNF; signaling via the Ret receptor tyrosine kinase) and Brain-derived neurotrophic factor (BDNF; signaling via the TrkB receptor tyrosine kinase) were reported to have protective and rescuing properties on DA neurons; however, their physiological roles in SN neurons remained unknown. Inactivation of the oxidative stress suppressor DJ-1 causes PD; remarkably, mice lacking DJ-1 function do not display overt SN degeneration, suggesting that additional DJ-1 interactors compensate for loss of DJ-1 function. To begin characterizing the cellular and molecular networks mediating SN neuron survival, I used mouse genetics to investigate the roles and the interaction between GDNF/BDNF-mediated trophic signaling and the DJ-1-mediated stress response in SN neurons. While mice lacking TrkB function specifically in SN neurons display a normal complement of SN neurons up to 24-months, loss of Ret function in DA neurons causes adult-onset and progressive SN degeneration, suggesting that GDNF/Ret signaling is required for long-term maintenance of SN neurons. I then generated and aged mice lacking Ret and DJ-1 and found remarkably that they display an enhanced SN degeneration relative to mice lacking Ret. Thus, DJ-1 promotes survival of Ret-deprived SN neurons. Interestingly, the survival requirement for Ret and DJ-1 is restricted to those SN neurons which express the ion channel GIRK2, project exclusively to the striatum and specifically degenerate in PD. This is the first in vivo evidence for a pro-survival role of DJ-1. To understand how DJ-1 interacts molecularly with Ret signaling, I performed epistasis analysis in Drosophila melanogaster. Although DJ-1 orthologs DJ-1A and DJ-1B are dispensable for fly development, the developmental defects induced by targeting constitutively active Ret to the retina were suppressed in a background of reduced DJ-1A/B function. Moreover, DJ-1A/B interacted genetically with Ras/ERK, but not PI3K/Akt signaling to regulate photoreceptor neuron development. Flies with reduced ERK activity and lacking DJ-1B function had more severe defects in photoreceptor neuron and wing development than flies with reduced ERK function. These observations establish, for the first time, a physiological role for DJ-1B in the intact Drosophila. Our findings suggest that the triple interaction between aging, trophic insufficiency and cellular stress may cause Parkinsonism. Because Ret and DJ-1 show convergence of their pro-survival activities, we predict that striatal delivery of GDNF might be most effective in PD patients carrying DJ-1 mutations. A better understanding of the molecular connections between trophic signaling, cellular stress and aging will accelerate the process of drug development in PD

Absence of Ret Signaling in Mice Causes Progressive and Late Degeneration of the Nigrostriatal System

Support of ageing neurons by endogenous neurotrophic factors such as glial cell line–derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration. GDNF has been tested in clinical trials for the treatment of Parkinson disease (PD), a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. BDNF modulates nigrostriatal functions and rescues DA neurons in PD animal models. The physiological roles of GDNF and BDNF signaling in the adult nigrostriatal DA system are unknown. We generated mice with regionally selective ablations of the genes encoding the receptors for GDNF (Ret) and BDNF (TrkB). We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation. These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.</p

Inactivation of VCP/ter94 Suppresses Retinal Pathology Caused by Misfolded Rhodopsin in Drosophila

The most common Rhodopsin (Rh) mutation associated with autosomal dominant retinitis pigmentosa (ADRP) in North America is the substitution of proline 23 by histidine (RhP23H). Unlike the wild-type Rh, mutant RhP23H exhibits folding defects and forms intracellular aggregates. The mechanisms responsible for the recognition and clearance of misfolded RhP23H and their relevance to photoreceptor neuron (PN) degeneration are poorly understood. Folding-deficient membrane proteins are subjected to Endoplasmic Reticulum (ER) quality control, and we have recently shown that RhP23H is a substrate of the ER–associated degradation (ERAD) effector VCP/ter94, a chaperone that extracts misfolded proteins from the ER (a process called retrotranslocation) and facilitates their proteasomal degradation. Here, we used Drosophila, in which Rh1P37H (the equivalent of mammalian RhP23H) is expressed in PNs, and found that the endogenous Rh1 is required for Rh1P37H toxicity. Genetic inactivation of VCP increased the levels of misfolded Rh1P37H and further activated the Ire1/Xbp1 ER stress pathway in the Rh1P37H retina. Despite this, Rh1P37H flies with decreased VCP function displayed a potent suppression of retinal degeneration and blindness, indicating that VCP activity promotes neurodegeneration in the Rh1P37H retina. Pharmacological treatment of Rh1P37H flies with the VCP/ERAD inhibitor Eeyarestatin I or with the proteasome inhibitor MG132 also led to a strong suppression of retinal degeneration. Collectively, our findings raise the possibility that excessive retrotranslocation and/or degradation of visual pigment is a primary cause of PN degeneration

Pro-Survival Role for Parkinson's Associated Gene DJ-1 Revealed in Trophically Impaired Dopaminergic Neurons

A mouse genetic study reveals a novel cell-survival role for the Parkinson's disease-associated gene DJ-1 in dopaminergic neurons that have reduced support from endogenous survival factors

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Progressive Loss of Nigral DA Neurons in <i>DAT-Ret^lx/lx</i> Mice

<div><p>(A) Coronal brain section of a 3-mo-old wild-type mouse showing DA neurons in the SNpc and the VTA stained with an antibody against TH. The inset shows a higher magnification view of the stippled area.</p> <p>(B and C) Stereological quantification of TH-positive DA neurons in the SNpc of 3-, 12-, and 24-mo-old control, <i>DAT-TrkB^lx/lx, DAT-Ret^lx/lx,</i> and double homozygous <i>Dat-Ret/TrkB</i> mice (C) (<i>n</i> = 3 mice per genotype), <i>Nes-Ret^lx/lx</i> mutant mice and littermate controls (D) (<i>n</i> = 4 mice per genotype). *, <i>p</i> < 0.05 (Student <i>t</i>-test).</p> <p>(D) Double immunostaining for NeuN and TH (very mild staining protocol to outline the SNpc [stippled area]). The inset shows a higher magnification view of the stippled box, displaying nuclear localization for NeuN and cytoplasmic immunoreactivity for TH.</p> <p>(E) Stereological quantification of NeuN-positive neurons in the SNpc of 12- and 24-mo-old control and <i>DAT-Ret^lx/lx</i> mice (<i>n</i> = 5 mice per genotype at 12 mo, and <i>n</i> = 4 mice per genotype at 24 mo). *, <i>p</i> < 0.0001 and <i>p</i> < 0.001 for 12- and 24-mo-old <i>DAT-Ret^lx/lx</i> mice, respectively (Student <i>t</i>-test).</p> <p>(F–H) Adjacent sections of SNpc and VTA of a 1-y-old wild-type mouse stained for TH (F), dopa-decarboxylase (G), and Pitx3 (H). Insets show higher magnification images.</p> <p>(I and J) Stereological quantification of DDC-positive (I) and Pitx3-positive (J) cells in the SNpc of 12-mo-old littermate control and <i>DAT-Ret^lx/lx</i> mice (<i>n</i> = 3 mice per genotype). *, <i>p</i> < 0.05 (Student <i>t</i>-test).</p> <p>(K and L) Stereological quantification of TH-positive cells in the VTA region of 1-y-old control and <i>DAT-Ret/TrkB</i> mutant mice (K) (<i>n</i> = 3 mice per genotype; <i>p</i> > 0.5; Student <i>t</i>-test) and in the LC of 12-mo-old control and <i>Nes-Ret^lx/lx</i> mutant mice (L) (<i>n</i> = 4 mice per genotype; <i>p</i> >0.5; Student <i>t</i>-test). Scale bar indicates 250 μm and, in insets, 100 μm.</p></div

Reduced Dopamine Release in the Striatum of <i>DAT-Ret^lx/lx</i> Mice

<div><p>(A) Total dopamine levels normalized to 2,3-dihydroxybenzoic acid (DHBA) and expressed relative to the weight of wet striatum (grams) of 2-y-old control mice <i>(Ret^lx/lx),</i> heterozygous <i>Ret^lx/−,</i> heterozygous <i>DAT-Ret^lx/+,</i> homozygous <i>DAT-Ret^lx/lx,</i> and <i>DAT-TrkB^lx/lx</i> mice. Note the minor reduction of total dopamine levels in all mice carrying the DAT-Cre knock-in construct.</p> <p>(B–E) Evoked dopamine release after electrical stimulation in the dorsal striatum of control mice (<i>Ret^lx/lx</i> and <i>Ret^lx/−</i> mice), heterozygous <i>DAT-Ret^lx/+</i> mice, and homozygous <i>DAT-Ret^lx/lx</i> mice of 1 y (B and C) or 2 y (D and E) of age. In both age groups, there is a significant decrease of released dopamine in the mice carrying the DAT-Cre knock-in construct compared to controls. There is a further significant decrease in the homozygous <i>DAT-Ret^lx/lx</i> mice due to the lack of Ret (<i>n</i> = 5 per genotype, <i>p</i> < 0.05, Student <i>t</i>-test). *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 (Student <i>t</i>-test). (C and E) Representative traces of single evoked dopamine release in different control and mutant mice.</p></div

Inflammation in SNpc of <i>DAT-Ret^lx/lx</i> Mice

<div><p>(A, B, D–I, K, and L) Immunohistochemical stainings of dorsal striatum (A and B) and SNpc (D–I, K, and L) of 24-mo-old control (A, D, E, H, and K) and <i>DAT-Ret^lx/lx</i> mice (B, F, G, I, and L) for Iba-1 (A, B, E, G, H, and I), TH (D and F), and MAC1 (K and L). To localize microglial cells in SNpc, adjacent sections were stained for TH, and the area of the SNpc was marked and copied to the adjacent section stained for macrophages.</p> <p>(C, J, and M) Histograms showing the number of Iba-1–positive (C and J) and MAC1-positive (M) cells in the striatum (C) and SNpc (J and M) of 24-mo-old (C and J) <i>DAT-Ret^lx/lx</i> mice and controls. No significant alterations in the numbers of Iba-1–positive cells were observed in the striatum of 24-mo-old mutants and controls ([C] <i>n</i> = 4, <i>p</i> = 0.065). A significant increase in the numbers of Iba-1–positive cells was observed in the SNpc of 24-mo-old <i>DAT-Ret^lx/lx</i> mice compared to controls (J) (<i>n</i> = 5, <i>p</i> < 0.05). The same result was obtained using MAC1 as a second independent microglial marker (M) (<i>n</i> = 3, <i>p</i> < 0.05). *, <i>p</i> < 0.05 (Student <i>t</i>-test). Scale bars indicate 100 μm.</p></div

Progressive Loss of Striatal Innervation in <i>DAT-Ret^lx/lx,</i> but Not <i>DAT-TrkB^lx/lx</i> Mice

<div><p>(A–D, F, and G) Representative images of dorsal striatum stained by immunofluorescence using antibodies against TH (A–D) and DAT (F and G) of control (A, B, and F) and <i>DAT-Ret^lx/lx</i> mutants (C, D, and G) at 12 (A, C, F, and G) and 24 mo of age (B and D).</p> <p>(E) The innervation density based on anti-TH immunofluorescence was quantified in dorsal versus ventral striatum of 12-mo-old controls (<i>n</i> = 16) versus <i>DAT-TrkB^lx/lx</i> (<i>n</i> = 4), <i>DAT-Ret^lx/lx</i> (<i>n</i> = 6), double <i>DAT-Ret/TrkB</i> (<i>n</i> = 5), and <i>Nes-Ret^lx/lx</i> mutants (<i>n</i> = 7). <i>DAT-Ret^lx/lx,</i> double <i>DAT-Ret/TrkB,</i> and <i>Nes-Ret^lx/lx</i> mutants showed significant reductions in TH fiber density in dorsal (<i>p</i> < 0.001) and ventral striatum (<i>p</i> < 0.001, <i>p</i> < 0.01, and <i>p</i> < 0.01 for <i>DAT-Ret^lx/lx,</i> double <i>DAT-Ret/TrkB,</i> and <i>Nes-Ret^lx/lx</i> mutants, respectively). **, <i>p</i> < 0.01 (Student <i>t</i>-test).</p> <p>(H) The innervation density based on anti-DAT immunofluorescence was quantified in 12-mo-old <i>Nes-Ret^lx/lx</i> mutants compared to age-matched controls (<i>n</i> = 4 per genotype; <i>p</i> < 0.001, Student <i>t</i>-test).</p> <p>(I) Time course of TH-positive fiber loss from 3 to 24 mo of age. <i>DAT-Ret^lx/lx</i> mutant mice show a progressive fiber loss, starting at 6 to 9 mo (<i>p</i> = 0.09 and <i>p</i> < 0.05 at 6 mo and 9 mo, respectively) and maximizing at 24 mo (<i>p</i> < 0.0001). <i>DAT-TrkB^lx/lx</i> mutant mice do not show any signs of fiber loss even at 24 mo of age (<i>p</i> = 0.13). *, <i>p</i> < 0.05; **, <i>p</i> < 0.01 (Student <i>t</i>-test). Scale bar indicates 25 μm.</p></div