Jack W. Lipton

Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines.

Rat progenitor cells from the germinal region of the fetal mesencephalon were isolated and expanded in media containing the mitogen epidermal growth factor. These cells remained mitotically active (up to 8 months), were immunoreactive for the progenitor cell marker nestin, and were readily infected with the BAG alpha retrovirus. When incubated in complete media containing serum in poly-L-lysine-coated plates, these cells spontaneously converted to neurons and glia but rarely expressed the dopamine (DA) neuron phenotype. Nineteen different cytokines were screened for their ability to induce the DA phenotype and only interleukin (IL)-1 was found to induce the expression of the DA neuron marker tyrosine hydroxylase (TH). The addition of IL-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) were found to further increase the number of TH immunoreactive (TH-ir) cells. The addition of mesencephalic membrane fragments and striatal culture-conditioned media along with the cytokine mixture induced the expression of morphologically mature TH-ir cells that were also immunoreactive for dopa-decarboxylase, the DA transporter, and DA itself. The DA neuron cell counts were approximately 20-25% of the overall cell population and 50% of the neurofilament population. Astrocytes and oligodendrocytes were also present. These data suggest that hematopoietic cytokines participate in the development of the DA neuron phenotype. Parallels between the function of hematopoietic cytokines in bone marrow and the central nervous system may exist and be useful in understanding the factors which regulate the differentiation of neurons in the brain.

Exposure to elevated levels of dietary fat attenuates psychostimulant reward and mesolimbic dopamine turnover in the rat

Recent studies indicate that decreased central dopamine is associated with diet-induced obesity in humans and in animal models. In the current study, the authors assessed the hypothesis that diet-induced obesity reduces mesolimbic dopamine function. Specifically, the authors compared dopamine turnover in this region between rats fed a high-fat diet and those consuming a standard low-fat diet. The authors also assessed behavioral consequences of diet-induced obesity by testing the response of these animals in a conditioned place paradigm using amphetamine as a reinforcer and in an operant conditioning paradigm using sucrose reinforcement. Results demonstrate that animals consuming a high-fat diet, independent of the development of obesity, exhibit decreased dopamine turnover in the mesolimbic system, reduced preference for an amphetamine cue, and attenuated operant responding for sucrose. The authors also observed that diet-induced obesity with a high-fat diet attenuated mesolimbic dopamine turnover in the nucleus accumbens. These data are consistent with recent hypotheses that the hormonal signals derived from adipose tissue regulate the activity of central nervous system structures involved in reward and motivation, which may have implications for the treatment of obesity and/or addiction.

IN UTERO BACTERIAL ENDOTOXIN EXPOSURE CAUSES LOSS OF TYROSINE HYDROXYLASE NEURONS IN THE POSTNATAL RAT MIDBRAIN

We investigated whether in utero exposure to the Gram(−) bacteriotoxin lipopolysaccharide (LPS) induces dopamine (DA) neuron loss in rats. The proinflammatory cytokine tumor necrosis factor α (TNF‐α) kills DA neurons and is elevated in the brains of patients with Parkinsons disease (PD). LPS is a potent inducer of TNF‐α, and both are increased in the chorioamniotic environment of women who have bacterial vaginosis (BV) during pregnancy, suggesting that BV might interfere with the normal development of fetal DA neurons. Gravid female rats were injected intraperitoneally with either LPS or normal saline at embryonic day 10.5 and their pups were killed at postnatal day 21. The brains of the pups were assessed for DA and TNF‐α levels and DA cell counts in the mesencephalon using tyrosine hydroxylase immunoreactive (THir) cells as a DA neuron marker. Prenatal LPS exposure significantly reduced striatal DA (29%) and increased DA activity (72%) as well as TNF‐α (101%). Stereological cell counts in the mesencephalon were also significantly reduced (27%) by prenatal LPS exposure. Prenatal exposure to LPS, as might occur in humans with BV, produces a significant loss of THir cells in rats that is still present 33 days following a single injection of LPS. Since this cell loss is well past the normal phase of DA neuron apoptosis that occurs in early postnatal life, rats so exposed may have a permanent loss of DA neurons, suggesting that prenatal infections may represent risk factors for PD.

Tumor Necrosis Factor α Is Toxic to Embryonic Mesencephalic Dopamine Neurons

Abstract Levels of the proinflammatory cytokine tumor necrosis factor α (TNFα) are increased in postmortem brain and cerebral spinal fluid from patients with Parkinsons disease (PD). This observation provides a basis for associating TNFα with neurodegeneration, but a specific toxicity in dopamine (DA) neurons has not been firmly established. Therefore, we investigated TNFα-induced toxicity in DA neurons by utilizing primary cultures of embryonic rat mesencephalon. Exposure to TNFα resulted in a dose-dependent decrease in DA neurons as evidenced by decreased numbers of tyrosine hydroxylase-immunoreactive (THir) cells. TNFα toxicity was selective for DA neurons in that neither glial cell counts nor the total number of neurons was decreased and no general cytotoxicity was evidenced by lactate dehydrogenase assay. Many of the cells which remained immunoreactive for TH had shrunken and rounded cell bodies with broken, blunted, or absent processes. However, TNFα-treated cultures also contained some THir cells which appeared to be undamaged and possibly resistant to TNFα-induced toxicity. Additionally, immunocytochemistry revealed basal expression of TNFα receptor 1 (p55, R1) and TNFα receptor 2 (p75, R2) on all cells within the mesencephalic cultures to some degree, even though only DA neurons were affected by TNFα treatment. These data strongly suggest that TNFα mediates cell death in a sensitive population of DA neurons and support the potential involvement of proinflammatory cytokines in the degeneration of DA neurons in PD.

Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-α, interleukin-1β, and nitric oxide

Parkinsons disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc). Although the exact mechanisms responsible for this cell loss are unclear, emerging evidence suggests the involvement of inflammatory events. In the present study, we characterized the effects of the proinflammatory bacteriotoxin lipopolysaccharide (LPS) on the number of tyrosine hydroxylase immunoreactive (THir) cells (used as an index for DA neurons) in primary mesencephalic cultures. LPS (10-80 microg/ml) selectively decreased THir cells and increased culture media levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well as nitrite (an index of nitric oxide (NO) production). Cultures exposed to both LPS and neutralizing antibodies to IL-1beta or TNF-alpha showed an attenuation of the LPS-induced THir cell loss by at least 50% in both cases. Inhibition of the inducible form of nitric oxide synthase (iNOS) by L-NIL did not affect LPS toxicity, but increased the LPS-induced levels of both TNF-alpha and IL-1beta. These findings suggest that neuroinflammatory stimuli which lead to elevations in cytokines may induce DA neuron cell loss in a NO-independent manner and contribute to PD pathogenesis.

Prenatal exposure to the bacteriotoxin lipopolysaccharide leads to long-term losses of dopamine neurons in offspring: a potential, new model of Parkinson's disease.

The cause of Parkinsons disease (PD) is currently unknown. Although a genetic cause has been implicated in familial PD, the vast majority of cases are considered idiopathic. Environmental toxins have been implicated as a cause for PD by many investigators. Unfortunately, the magnitude of this exposure would likely need to be very high and as a result, would likely have been identified by the many epidemiological studies performed to date. Recently, we inadvertently realized that exposure to neurotoxins while still in utero may also represent a risk factor. Thus, exposure to the bacteriotoxin, lipopolysaccharide (LPS) during a critical developmental window in rats, leads to the birth of animals with fewer than normal dopamine (DA) neurons. This DA neuron loss is apparently permanent as it is still present in 16 months old animals (the longest period studied to date). Moreover, the loss of DA neurons seen in these animals increases with age thereby mimicking the progressive pattern of cell loss seen in human PD. The DA neuron loss is accompanied by reductions in striatal DA, increases in DA activity, and increased production of the pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). These are also characteristics of the PD brain. This model therefore shares many of the same characteristics with PD, and most importantly exhibits a slow, protracted loss of DA neurons - a characteristics of this animal model not found in other models. Interestingly, a common complication of pregnancy is a condition known as bacterial vaginosis (BV), which is known to produce increased levels of LPS and pro-inflammatory cytokines in the chorioamniotic environment of the fetus. This raises the interesting possibility that BV may be a risk factor for PD. The possibility that prenatal toxin exposure may contribute to the development of a neurodegenerative disease of the aged raises interesting new pathogenic questions and draws attention to the possibility that in utero exposure to neurotoxins may represent a here to fore unrecognized cause of PD.

Leptin Regulates Energy Balance and Motivation Through Action at Distinct Neural Circuits

BACKGROUND Overconsumption of calorically dense foods contributes substantially to the current obesity epidemic. The adiposity hormone leptin has been identified as a potential modulator of reward-induced feeding. The current study asked whether leptin signaling within the lateral hypothalamus (LH) and midbrain is involved in effort-based responding for food rewards and/or the modulation of mesolimbic dopamine. METHODS The contribution of endogenous leptin signaling for food motivation and mesolimbic dopamine tone was examined after viral-mediated reduction of the leptin receptor within LH and midbrain neurons in male rats. RESULTS Knockdown of leptin receptors selectively in the LH caused increased body weight, caloric consumption, and body fat in rats maintained on a calorically dense diet. Knockdown of leptin receptors selectively in midbrain augmented progressive ratio responding for sucrose and restored high-fat, diet-induced suppression of dopamine content in the nucleus accumbens. CONCLUSIONS In summary, endogenous leptin signaling in the hypothalamus restrains the overconsumption of calorically dense foods and the consequent increase in body mass, whereas leptin action in the midbrain regulates effort-based responding for food rewards and mesolimbic dopamine tone. These data highlight the ability of leptin to regulate overconsumption of palatable foods and food motivation through pathways that mediate energy homeostasis and reward, respectively.

Combined toxicity of prenatal bacterial endotoxin exposure and postnatal 6-hydroxydopamine in the adult rat midbrain

We previously reported that injection of the Gram (-) bacteriotoxin, lipopolysaccharide (LPS), into gravid females at embryonic day 10.5 led to the birth of animals with fewer than normal dopamine (DA) neurons when assessed at postnatal days (P) 10 and 21. To determine if these changes continued into adulthood, we have now assessed animals at P120. As part of the previous studies, we also observed that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) was elevated in the striatum, suggesting that these animals would be more susceptible to subsequent DA neurotoxin exposure. In order to test this hypothesis, we injected (at P99) 6-hydroxydopamine (6OHDA) or saline into animals exposed to LPS or saline prenatally. The results showed that animals exposed to prenatal LPS or postnatal 6OHDA alone had 33% and 46%, respectively, fewer DA neurons than controls, while the two toxins combined produced a less than additive 62% loss. Alterations in striatal DA were similar to, and significantly correlated with (r(2)=0.833) the DA cell losses. Prenatal LPS produced a 31% increase in striatal TNFalpha, and combined exposure with 6OHDA led to an 82% increase. We conclude that prenatal exposure to LPS produces a long-lived THir cell loss that is accompanied by an inflammatory state that leads to further DA neuron loss following subsequent neurotoxin exposure. The results suggest that individuals exposed to LPS prenatally, as might occur had their mother had bacterial vaginosis, would be at increased risk for Parkinsons disease.

Diaphragm Pacing with a Quadripolar Phrenic Nerve Electrode: An International Study

We sought to determine the international experience with the quadripolar diaphragm pacer system and to test two hypotheses: the incidence of pacer complications would be (1) increased among pediatric as compared to adult patients; and (2) highest among active pediatric patients with idiopathic congenital central hypoventilation syndrome (CCHS). Data were collected via a questionnaire coupled with the Atrotech Registry data for a total of 64 patients (35 children and 29 adults) from 14 countries. Thoracic implantation of electrodes and bilateral pacer use each occurred in 94% of all subjects. Tetraplegic (vs pediatric CCHS) patients were more typically paced 24 hours/day (P = 0.001). Pacing duration averaged 2.0 ± 1.0 years among children and 2.2 ± 1.1 years among adults. Infections occurred among 2.9% of surgical procedures, all in pediatric CCHS patients (vs pediatric tetraplegic patients, P = 0.01). The incidence of mechanical trauma was 3.8%, without significant differences among patient groups. The incidence of presumed electrode and receiver failure were 3.1% and 5.9%, respectively, with internal component failure greater among pediatric CCHS than pediatric tetraplegic patients (P < 0.01). Intermittent or absent function of 0–4 electrode combinations occurred among 19% of all patients, with increased frequency among pediatric CCHS than pediatric tetraplegic patients (P < 0.03). Complication‐ free successful pacing occurred in 60% of pediatric and 52% of adult patients. In all, 94 % of the pediatric and 86% of the adult patients paced successfully after the necessary intervention. Although pacer complications were not increased among pediatric as compared to adult patients, the incidence of complications was highest among the active pediatric patients with CCHS. Longitudinal study of these patients will provide invaluable information for modification and improvement of the quadripolar system.

Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinsons disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague-Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting that PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time-point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.