What determines resilience in patients with Parkinson's disease?

OBJECTIVE: To investigate the relationship of resilience to disease severity, disability, quality of life (QoL) and non-motor symptoms in Parkinson's disease (PD). A secondary objective was to investigate whether resilience is distinct from other personality domains in PD. BACKGROUND: Resilience is the ability to reestablish emotional equilibrium in the face of adversity. It may play a pivotal role in disability and quality of life and has not been studied in PD. METHODS: 83 PD patients (Age 66.3 ± 10.6, Total Unified Parkinson's Disease Rating Scale (T-UPDRS) 36.9 ± 17.8) completed the Resilience Scale 15 (RS-15). Scales measuring disability, mental and physical health-related QoL, non-motor symptoms (depression, anxiety, somatization, apathy, fatigue), and personality domains were completed. Pearson's correlations were analyzed between these scales and the RS-15. RESULTS: Greater resilience correlated with less disability (r = -.30, p = .01), and better physical and mental QoL (r = .31, p < .01; r = .29, p = .01), but not with PD severity (T-UPDRS, r = -.17, p > .05). Among non-motor symptoms and personality domains, resilience strongly correlated with less apathy (r = -.66), less depression (r = -.49), and more optimism (r = .54, all p < .001). Moderate correlations were seen between more resilience, reduced fatigue (r = -.40) and anxiety (r = -.34; both p < .001). CONCLUSIONS: Resilience correlated with less disability and better QoL but not with PD severity. Resilience was also highly associated with both non-motor symptoms (less apathy, depression, fatigue) and a personality domain (more optimism). The role of resilience in helping patients adapt to living with symptoms of chronic disease may explain its lack of correlation with PD severity.

When do patients with Parkinson disease disclose their diagnosis?

The authors surveyed 101 patients with Parkinson disease (PD) about their experiences disclosing the diagnosis. Ninety percent disclosed early to family; more than 25% waited at least 1 year to disclose at work. The main concerns about disclosure were fear of reflecting negatively on themselves and fear of upsetting others. Patients who delayed disclosure were more likely male, younger, and employed. There is considerable variability among patients with PD in the time to disclose their diagnosis.

Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS.

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.

Glutamate-pyruvate transaminase protects against glutamate toxicity in hippocampal slices.

Elimination of glutamate through enzymatic degradation is an alternative to glutamate receptor blockade in preventing excitotoxic neuronal injury. Glutamate pyruvate transaminase (GPT) is a highly active glutamate degrading enzyme that requires pyruvate as a co-substrate. This study examined the ability of GPT to protect neurons of the hippocampal slice preparation against glutamate toxicity. Two methods were used to elevate the concentration of glutamate in the peri-neuronal space. In an endogenous release paradigm, slices were incubated with 100-500 microM L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate re-uptake. One hour of exposure to PDC in normal, pyruvate-free slice maintenance medium caused a dose dependent increase in neuronal death assessed 24 h later by propidium iodide uptake in dead cell nuclei. GPT (10 U/ml) decreased neuronal death caused by exposure to PDC at all PDC concentrations tested. Neuroprotection in this model was not dependent on added or non-physiologic levels of pyruvate. In a different paradigm, glutamate was added directly to the normal, pyruvate-free slice maintenance medium and not rinsed away, exposing the slices to a range of 1-5 mM glutamate for an extended period. Twenty-four hours later, neuronal death was again assessed by propidium iodide uptake. GPT was again neuroprotective, decreasing neuronal death in the range from 3 to 5 mM glutamate. In the setting of incubation with this large load of glutamate, neuroprotection by GPT was enhanced by adding pyruvate to the medium. GPT is an effective neuroprotectant against glutamate excitotoxicity. When exposure is limited to endogenously released glutamate, neuroprotection by GPT is not dependent on added pyruvate.

Interaction of tetanus toxin derived hybrid proteins with neuronal cells.

The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E. coli. Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry. Hybrid proteins were bound and internalized in a manner very similar to TTC. Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types. Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization. Proteins coupled to TTC share its long-term stability after cellular internalization. After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol. TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.

Protective effect of supplemental superoxide dismutase on survival of neuronal cells during starvation. Requirement for cytosolic distribution.

There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation. Cells incubated with TTC-hSOD1 showed a threefold increase in cellular SOD activity over control cells. This level of increase was comparable to fetal cortical neurons from transgenic mice constitutively expressing hSOD1 and transfected N18-RE-105 cells expressing a green fluorescent protein-hSOD1 fusion protein (GFP-hSOD1). Human SOD1 was distributed diffusely throughout the cytoplasm of the transgenic murine neurons and transfected N18-RE-105 cells. In contrast, cells incubated with TTC-hSOD1 showed hSOD1 localized to the cell surface and intra-cytoplasmic vesicles. The cells expressing hSOD1 showed enhanced survival in glucose- and pyruvate-free medium. Neither cortical neurons nor N18-RE-105 cells incubated in TTC-hSOD1 showed increased survival during starvation. Access to the site where toxic superoxides are generated or their targets may be necessary for the protective function of SOD1.

Enzymatic degradation protects neurons from glutamate excitotoxicity.

Several enzymes with the capacity to degrade glutamate have been suggested as possible neuroprotectants. We initially evaluated the kinetic properties of glutamate pyruvate transaminase (GPT; also known as alanine aminotransferase), glutamine synthetase, and glutamate dehydrogenase under physiologic conditions to degrade neurotoxic concentrations of glutamate. Although all three enzymes initially degraded glutamate rapidly, only GPT was able to reduce toxic (500 microM) levels of glutamate into the physiologic (<20 microM) range. Primary cultures of fetal murine cortical neurons were subjected to paradigms of either exogenous or endogenous glutamate toxicity to evaluate the neuroprotective value of GPT. Neuronal survival after exposure to added glutamate ranging from 100 to 500 microM was improved significantly in the presence of GPT (> or =1 U/ml). Cultures were also exposed to the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), which produces neuronal injury by elevating extracellular glutamate. GPT significantly reduced the toxicity of PDC. This reduction was associated with a reduction in the PDC-dependent rise in the medium concentration of glutamate. These results suggest that enzymatic degradation of glutamate by GPT can be an alternative to glutamate receptor blockade as a strategy to protect neurons from excitotoxic injury.

Enhancement of diphtheria toxin potency by replacement of the receptor binding domain with tetanus toxin C-fragment: a potential vector for delivering heterologous proteins to neurons.

This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB(389)TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB(389)) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB(389)TTC was approximately 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB(389)MSH. The cytotoxic effect of DAB(389)TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB(389)TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB(389) catalytic fragment. These results suggest that a catalytically inactive form of DAB(389)TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.

alpha 1-tubulin expression in proximally axotomized mouse cortical neurons.

This paper further characterizes the response to axotomy of mouse transcallosal cortical neurons, a population of neurons that seems to be particularly refractory to regeneration. Mouse transcallosal cortical neurons did not upregulate mRNA for the growth-associated protein alpha 1-tubulin following axotomy, even when the axonal distance from injury to cell body was only 100-300 microns. Previous experiments had found no upregulation of another growth-associated protein, GAP-43, by transcallosal neurons following axotomy 1-2 mm from the cell body. These latest results establish that this population of neurons fails to respond to axotomy even when it is extremely proximal and that this failure is not a peculiarity specific to one growth-associated protein but is indicative of a generally poor regenerative response.

Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytes.

The formation of functional synapses is a late milestone of neuronal differentiation. The establishment of functional synapses can be used to assess neuronal characteristics of different cell lines. In the present study, we examined the in vitro conditions that influence the ability of human neurons derived from the NT2 cell line (NT2N neurons) to establish synapses. The morphologic, immunologic, and electrophysiologic characteristics of these synapses was examined. In the absence of astrocytes, NT2N neurons rarely formed synapses and their action potentials were weak and uncommon. In contrast, when plated on primary astrocytes, NT2N neurons were able to form both glutamatergic excitatory (71%) and GABAergic inhibitory (29%) functional synapses whose properties (kinetics, ion selectivity, pharmacology, and ultrastructure) were similar to those of synapses of neurons in primary cultures. In addition, coculture of NT2N neurons with astrocytes modified the morphology of the neurons and extended their in vitro viability to more than 1 year. Because astrocyte-conditioned medium did not produce these effects, we infer that direct contact between NT2N neurons and astrocytes is required. These results suggest that NT2N neurons are similar to primary neurons in their synaptogenesis and their requirement for glial support for optimal survival and maturation. This system provides a model for further investigations into the neurobiology of synapses formed by human neurons.

Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (H(c)).

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.

Death of transcallosal neurons after close axotomy.

The extent of cell death after axotomy may limit potential recovery after brain injury. We wished to determine the effect of axotomizing lesions on survival of transcallosally projecting cortical neurons. Transcallosal neurons were prelabeled by retrograde transport of the fluorescent dyes Fluoro-Gold and True Blue. A transcortical stab wound divided the field of labeled cortical cells into axotomized and unaxotomized groups. Little difference in labeled cell density was seen over the first few days after injury. Animals surviving at least 2 weeks after injury had clear loss of axotomized neurons. By 1 month after injury, the vast majority of axotomized labeled cells appeared to have died. Quantitative evaluation of labeled cells showed that the region of cortex within 1 mm of the axotomizing injury had less than 10% of the expected neuronal density in animals surviving at least 4 weeks after injury. Close axotomy appears to cause dramatic loss of transcallosal neurons even in adult animals.

Delivery of recombinant tetanus-superoxide dismutase proteins to central nervous system neurons by retrograde axonal transport.

The nontoxic C fragment of tetanus toxin (TC) can transport other proteins from the circulation to central nervous system (CNS) motor neurons. Increased levels of CuZn superoxide dismutase (SOD) are protective in experimental models of stroke and Parkinson's disease, whereas mutations in SOD can cause motor neuron disease. We have linked TC to SOD and purified the active recombinant proteins in both the TC-SOD and SOD-TC orientations. Light microscopic immunohistochemistry and quantitative enzyme-linked immunosorbant assays (ELISA) of mouse brainstem, after intramuscular injection, demonstrate that the fusion proteins undergo retrograde axonal transport and transsynaptic transfer as efficiently as TC alone.

Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse.

As an approach to understanding why central neurons fail to regenerate, we have studied the response to proximal axotomy of transcallosal neurons of the cerebral cortex of the mouse. Anatomical studies have indicated only very slight regenerative responses by this population of cortical neurons. To further examine the regenerative response of these cells, we have looked by in situ hybridization at the expression of GAP-43 mRNA following axotomy caused by a stab wound delivered within about 200 microm to 1.25 mm of the cell body. Axotomized transcallosal neurons were compared with near-by unaxotomized transcallosal neurons, as well as with distant unaxotomized cortical neurons in the contralateral hemisphere. All three populations of neurons had been pre-labeled with Fluoro-Gold to allow identification. No up-regulation of GAP-43 mRNA above background levels was detected for axotomized cortical neurons at 1, 3 or 7 days after injury. In contrast, increases in mean silver grain density of up to 8-fold were measured in axotomized spinal cord motor neurons used as positive controls. Thus, as a population, the transcallosal cortical pyramidal neurons did not show a significant regenerative response, as monitored by GAP-43 upregulation, even with very close axotomy. These results identify this population of neurons as among the least regenerative studied, and suggest that, on a molecular level, inherent neuronal properties play a role in the limited regenerative response to brain injury.

Free-living daily energy expenditure in patients with Parkinson's disease.

Previous studies have suggested that elevated resting energy expenditure contributes to weight loss in patients with Parkinson's disease (PD). Body weight is, however, ultimately determined by variation in daily energy expenditure and not just resting energy expenditure. Therefore, we examined the hypothesis that PD patients are characterized by elevated daily energy expenditure. Sixteen patients with levodopa responsive PD and 46 healthy elderly controls were characterized for daily energy expenditure and its components (resting and physical activity energy expenditure) using a combination of the doubly labeled water technique (over 10 days) and resting indirect calorimetry. Fat-free mass and fat mass were measured by dual energy x-ray absorptiometry. Results showed that fat mass and fat-free mass did not differ between groups. Daily energy expenditure was 15% lower (2214 +/- 460 vs. 2590 +/- 497 kcal/d; p < 0.01) in PD patients compared to controls. This was primarily due to lower physical activity energy expenditure (339 +/- 366 vs. 769 +/- 412 kcal/d; P < 0.01) in PD patients as resting energy expenditure was not different between groups (1655 +/- 283 vs. 1561 +/- 219 kcal/d). These results show that daily energy expenditure is lower in PD patients compared to healthy elderly, primarily due to reduced physical activity energy expenditure. These results argue against the hypothesis that an abnormally elevated daily energy expenditure contributes to weight loss in PD.

Sarcopenia in aging humans: the impact of menopause and disease.

We examine the association of the menopause transition, congestive heart failure, and Parkinson's disease on body composition and energy expenditure. We present evidence suggesting that the normal menopausal transition is associated with accelerated loss of fat-free mass, a decline in resting metabolic rate, and increased central body fatness. Second, we show that the cardiac cachexia associated with heart failure is partially due to an elevated level of energy expenditure. Despite having a lower quantity of fat-free mass, congestive heart failure patients have a higher resting metabolic rate (approximately 283 kcal/d) for their metabolic size than healthy elderly. The elevated level of resting energy expenditure probably contributes to their unexplained weight loss. Parkinson's patients experience muscular rigidity and tremor which could contribute to inappropriately high levels of energy expenditure and difficulty in maintaining body weight and composition. We examined resting metabolic rate and body composition in eight Parkinson's patients and 34 healthy age-matched controls. Parkinson's patients showed lower levels of fat-free mass (approximately 6 kg), but similar resting metabolic rates (1601 +/- 250 kcal/d) versus healthy controls (1671 +/- 212 kcal/d), suggesting a hypermetabolic state. A re-examination of daily energy needs and the metabolic factors contributing to periods of energy imbalance during the menopausal transition and in several disease states may be a prerequisite to offsetting accelerated sarcopenia.

Internalization of IgG in motoneurons of patients with ALS: selective or nonselective?

The hypothesis that abnormal antibodies may be involved in the pathogenesis of ALS has been supported in part by IgG's being present within motoneurons of ALS patients more frequently than in motoneurons of controls. IgG, as well as other serum proteins, is also present in motoneurons of normal human and animal spinal cords. We attempted to determine whether the IgG found in motoneurons of ALS patients was localized by an immune-specific or nonspecific process. To address this question, we used immunocytochemistry to evaluate the presence and relative density of different serum proteins in spinal cords from nine patients with ALS. Both IgG and alpha 2-macroglobulin (alpha 2Mac) were present in motoneurons in all nine cases. More important, there was a close concordance between the IgG and alpha 2Mac immunolabeling of motoneurons. The presence of a nonimmune plasma protein--alpha 2Mac--in a similar distribution to IgG and with a similar intensity implies that the internalization of these proteins in motoneurons of patients with ALS is best explained by a nonselective mechanism of endocytosis of extracellular fluid.

CuZn superoxide dismutase (SOD-1):tetanus toxin fragment C hybrid protein for targeted delivery of SOD-1 to neuronal cells.

Increased levels of CuZn superoxide dismutase (SOD-1) are cytoprotective in experimental models of neurological disorders associated with free radical toxicity (e.g. stroke, trauma). Targeted delivery of SOD-1 to central nervous system neurons may therefore be therapeutic in such diseases. The nontoxic C-fragment of tetanus toxin (TTC) possesses the nerve cell binding/transport properties of tetanus holotoxin and has been used as a vector to enhance the neuronal uptake of proteins including enzymes. We have now produced a recombinant, hybrid protein in Escherichia coli tandemly joining human SOD-1 to TTC. The expressed hybrid protein (SOD:Tet450) has a subunit molecular mass of 68 kDa and is recognized by both anti-SOD-1 and anti-TTC antibodies. Calculated per mol, SOD:Tet450 has approximately 60% of the expected SOD-1 enzymatic activity. Analysis of the hybrid protein's interaction with the neuron-like cell line, N18-RE-105, and cultured hippocampal neurons by enzyme immunoassay for human SOD-1 revealed that SOD:Tet451 association with cells was neuron-specific and dose-dependent. The hybrid protein was also internalized, but there was substantial loss of internalized hybrid protein over the first 24 h. Hybrid protein associated with cells remained enzymatically active. These results suggest that human SOD-1 and TTC retain their respective functional properties when expressed together as a single peptide. SOD:Tet451 may prove to be a useful agent for the targeted delivery of SOD-1 to neurons.

CNS gene delivery by retrograde transport of recombinant replication-defective adenoviruses.

The ability to program recombinant gene expression in specific sets of motor and sensory neurons would facilitate the treatment of a number of acquired and inherited central nervous system (CNS) diseases. In this report, we demonstrate that intramuscular injection of replication-defective recombinant adenovirus results in high-level recombinant gene expression, specifically in the CNS motor and sensory neurons that innervate the inoculated muscles. Neural expression of the recombinant genes results from virus transport into the CNS, presumably by retrograde axonal transport. This novel method of neural gene delivery may be of value in studies designed to improve understanding and treatment of inherited and acquired neurological diseases.