Quantitative morphological comparison of axon-targeting strategies for gene therapies directed to the nigro-striatal projection.

Cellular targeting of mRNAs and proteins to axons is essential for axon growth during development and is likely to be important for adult maintenance as well. Given the importance and potency of these axon-targeting motifs to the biology of axons, it seems possible that they can be used in the design of transgenes that are intended to enhance axon growth or maintenance, so as to improve potency and minimize off-target effects. To investigate this possibility, it is first essential to assess known motifs for their efficacy. We have therefore evaluated four axon-targeting motifs, using adeno-associated viral vector-mediated gene delivery in the nigro-striatal dopaminergic system, a projection that is predominantly affected in Parkinson's disease. We have tested two mRNA axonal zipcodes, the 3' untranslated region (UTR) of ß-actin and 3' UTR of tau, and two axonal-targeting protein motifs, the palmitoylation signal sequence in GAP-43 and the last 15 amino acids in the amyloid precursor protein, to direct the expression of the fluorescent protein Tomato in axons. These sequences, fused to Tomato, were able to target its expression to dopaminergic axons. Based on quantification of Tomato-positive axons, and the density of striatal innervation, we conclude that the C-terminal of the amyloid precursor protein is the most effective axon-targeting motif.

GDNF as a candidate striatal target-derived neurotrophic factor for the development of substantia nigra dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) has been known for many years to protect and restore dopamine neurons of the substantia nigra (SN) in lesion models of parkinsonism, but much less has been known of its normal physiologic role. We have found that GDNF injected into the striatum postnatally suppresses naturally-occurring cell death in SN dopamine neurons, and neutralizing antibodies augments it. Neutralizing antibodies augment cell death during the first phase, which occurs during the first postnatal week, but not during the second phase in the second week. To further explore the possible neurotrophic role of GDNF, we created double transgenic mice which overexpress GDNF exclusively in the target regions of mesencephalic neurons, particularly the striatum. As anticipated for a limiting, target-derived factor, this resulted in an increased surviving number of SN dopamine neurons after the first phase of cell death. However, this increase did not persist into adulthood. We conclude that GDNF is the leading candidate for a target-derived neurotrophic factor for SN dopamine neurons during the first phase of cell death, but that other factors must play an essential role in later development.

The expression of proapoptosis genes is increased in bipolar disorder, but not in schizophrenia.

Post-mortem studies conducted over the past 15 years suggest that apoptosis could play a role in the pathophysiology of bipolar disorder (BD) and, to a lesser degree, schizophrenia (SZ). To test this hypothesis, we have performed a post hoc analysis of an extant gene expression profiling database obtained from the hippocampus using a novel methodology with improved sensitivity. Consistent with the working hypothesis, BDs showed a marked upregulation of 19 out of 44 apoptosis genes; however, contrary to the hypothesis, the SZ group showed a downregulation of genes associated with apoptotic injury and death. These changes in the regulation of apoptosis genes were validated using quantitative RT-PCR. Additionally, antioxidant genes showed a marked downregulation in BDs, suggesting that accumulation of free radicals might occur in the setting of a previously reported decrease of the electron transport chain in this disorder. Overall, the changes seen in BDs and SZs do not appear to be related to exposure to either neuroleptics or mood stabilizers. We conclude that fundamental differences in the genetic regulation of apoptosis and antioxidant genes may help discriminate between the pathophysiology of BD and SZ and potentially point to new treatment strategies that are specific for each disorder.

Mapping of hippocampal gene clusters regulated by the amygdala to nonlinkage sites for schizophrenia.

A recent study using a 'partial' rodent model of schizophrenia has employed amygdalar activation to induce reported changes in the expression of hippocampal genes associated with metabolic and signaling pathways in response to amygdalar activation. The amygdalo-hippocampal pathway plays a central role in the regulation of the stress response and emotional learning. In the current study, we have performed a chromosome mapping analysis to determine whether genes showing changes in response to environmental stress may form clusters and, if so, whether they might show a topographical association with linkage sites for schizophrenia. When the hippocampal genes showing changes in expression were topographically mapped on specific rat chromosomes, significant clustering was observed on chromosomes 1, 4 and 8, although chromosome 1 showed the largest amount of clustering. When these same rodent genes were mapped to human chromosomes, most of the genes found on chromosome 1 in rat mapped to chromosome 11 in human. The vast majority of the genes showing changes in regulation were excluded from known linkage sites for schizophrenia. Based on these findings, we postulate that environmental factors may contribute to the endophenotype for schizophrenia through the activation and/or deactivation of specific genetic clusters, ones that do not appear to be directly associated with susceptibility genes for this disorder.

Acute amygdalar activation induces an upregulation of multiple monoamine G protein coupled pathways in rat hippocampus.

A "partial" rodent model for schizophrenia has been used to characterize the regulation of hippocampal genes in response to amygdalar activation. At 96 h after the administration of picrotoxin into the basolateral nucleus, we have observed an increase in the expression of genes associated with 18 different monoamine (ie adrenergic alpha 1, alpha 2 and beta 2, serotonergic 5HT5b and 5HT6, dopamine D4 and muscarinic m1, m2 and m3) and peptide (CCK A and B, angiotensin 1A, mu and kappa opiate, FSH, TSH, LH, GNRH, and neuropeptide Y) G-protein coupled receptors (GPCRs). These latter receptors are associated with three different G protein signaling pathways (Gq, Gs, and Gi) in which significant changes in gene expression were also noted for adenylate cyclase (AC4), phosphodiesterase (PDE4D), protein kinase A (PKA), and protein kinase C (PKC). Quantitative RT-PCR was used to validate the results and demonstrated that there were predictable increases of three GPCRs selected for this analysis, including the dopamine D4, alpha 1b, and CCK-B receptors. Eight out of the nine monoamine receptors showing these changes have moderate to high affinity for the atypical antipsychotic, clozapine. Taken together, these results suggest that amygdalar activation may play a role in the pathophysiology and treatment of psychosis by regulating the activity of multiple GPCR and metabolic pathways in hippocampal cells.

Spindle model responsive to mixed fusimotor inputs and testable predictions of beta feedback effects.

Skeletofusimotor (beta) motoneurons innervate both extrafusal muscle units and muscle fibers within muscle spindle stretch receptors. By receiving excitation from group Ia muscle spindle afferents and driving the muscle spindle afferents that excite them, they form a positive feedback loop of unknown function. To study it, we developed a computationally efficient model of group Ia afferent behavior, capable of responding to multiple fusimotor inputs, that matched experimental data. This spindle model was then incorporated into a simulation of group Ia feedback during ramp/hold and triangular stretches with and without closure of the beta loop, assuming that gamma and beta fusimotor drives of the same type (static or dynamic) have identical effects on spindle afferent firing. The effects of beta feedback were implemented by driving a fusimotor input with a delayed and filtered fraction of the spindle afferent output. During triangular stretches, feedback through static beta motoneurons enhanced Ia afferent firing during shortening of the spindle. In contrast, closure of a dynamic beta loop increased Ia firing during lengthening. The strength of beta feedback, estimated as a "loop gain" was comparable to experimental estimates. The loop gain increased with velocity and amplitude of stretch but decreased with increased superimposed gamma fusimotor rates. The strongest loop gains were seen when the beta loop and the gamma bias were of different types (static vs. dynamic).

Developmental changes in short-term synaptic depression in the neonatal mouse spinal cord.

We examined age-dependent changes in short-term synaptic depression of monosynaptic excitatory postsynaptic potentials (EPSPs) recorded in lumbar motoneurons in hemisected spinal cords of neonatal Swiss-Webster mice between postnatal day 2 (P2) and 12 (P12). We used four paradigms that sample the input-output dependence on stimulation history in different but complementary ways: 1) paired-pulse depression; 2) steady-state depression during constant frequency trains; 3) modulation during irregular stimulation sequences; and 4) recovery after high-frequency conditioning trains. Paired-pulse synaptic depression declined more than steady-state depression during 10-pulse trains at frequencies from 0.125 to 8 Hz in this age range. Depression during sequences of irregular stimulations that more closely mimic physiological activation also declined with postnatal age. On the other hand, the overall rate of synaptic recovery after a 4-Hz conditioning train exhibited surprisingly little change between P2 and P12. Control experiments indicated that these observations depend primarily, if not exclusively, on changes in presynaptic transmitter release. The data were examined using quantitative models that incorporate factors that have been suggested to exist at more specialized central synapses. The model that best predicted the observations included two presynaptic compartments that are depleted during activation, plus two superimposed processes that enhance transmitter release by different mechanisms. One of the latter produced rapidly-decaying enhancement of transmitter release fraction. The other mechanism indirectly enhanced the rate of renewal of one of the depleted presynaptic compartments. This model successfully predicted the constant frequency and irregular sequence data from all age groups, as well as the recovery curves following short, high-frequency tetani. The results suggest that a reduction in release fraction accounts for much of the decline in synaptic depression during early postnatal development, although changes in both enhancement processes also contribute. The time constants of resource renewal showed surprisingly little change through the first 12 days of postnatal life.

The expression of mRNAs for the proteasome complex is developmentally regulated in the rat mesencephalon.

The proteasome is a large protease complex that recognizes, unfolds and degrades ubiquitinated proteins. Evidence is now accumulating that the ubiquitin-proteasome system may play an important role in neuronal apoptosis. However, little is known about the involvement of the proteasome in neuronal death in vivo, and there has been no prior analysis of the developmental expression of proteasome subunits in brain during periods of natural and inducible apoptotic death. We therefore studied the mRNA expression levels, using Northern analysis, of a subunit from each of the three key components of the proteasome in the rat mesencephalon from E21 through development and in adulthood. We measured mRNA expression for RC6 (a subunit of 20S), p112 (a subunit of 19S) and PA28-alpha (a subunit of 11S). The expression of PA28-alpha in rat mesencephalon was highest at the earliest times studied, and then decreased at PND 21, 28 and adult, in comparison to E21 (P<0.05) and PND 2, 4 and 7 (P<0.01). The expression of RC6 was lower in adult in comparison to PND 2, 4 and 21 (P<0.05) and PND 14 (P<0.01). There were no significant differences in the mRNA levels of p112 at various times studied. In situ hybridization at PND 7 indicated that all the subunits studied are particularly abundant in the SNpc. Thus, PA28-alpha and RC6 are developmentally regulated, and they may therefore play a role in developmental cell death or differentiation in neurons of the SN.

Patterns of locomotor drive to motoneurons and last-order interneurons: clues to the structure of the CPG.

We have examined the linkage between patterns of activity in several hindlimb motor pools and the modulation of oligosynaptic cutaneous reflex pathways during fictive locomotion in decerebrate unanesthetized cats to assess the notion that such linkages can shed light on the structure of the central pattern generator (CPG) for locomotion. We have concentrated attention on the cutaneous reflex pathways that project to the flexor digitorum longus (FDL) motor pool because of that muscle's unique variable behavior during normal and fictive locomotion in the cat. Differential locomotor control of last-order excitatory interneurons in pathways from low-threshold cutaneous afferents in the superficial peroneal and medial plantar afferents to FDL motoneurons is fully documented for the first time. The qualitative patterns of differential control are shown to remain the same whether the FDL muscle is active in early flexion, as usually found, or during the extension phase of fictive locomotion, which is less common during fictive stepping. The patterns of motor pool activity and of reflex pathway modulation indicate that the flexion phase of fictive locomotion has distinct early versus late components. Observations during "normal" and unusual patterns of fictive stepping suggest that some aspects of locomotor pattern formation can be separated from rhythm generation, implying that these two CPG functions may be embodied, at least in part, in distinct neural organizations. The results are discussed in relation to a provisional circuit diagram that could explain the experimental findings.

Expression of cyclin-dependent kinase 5 and its activator p35 in models of induced apoptotic death in neurons of the substantia nigra in vivo.

Cyclin-dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6-hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co-labeling with a neuron-specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.

Short-term synaptic depression in the neonatal mouse spinal cord: effects of calcium and temperature.

We have studied short-term synaptic depression of excitatory postsynaptic potentials (EPSPs) in lumbosacral motoneurons in the isolated, in vitro spinal cord of neonatal mice at 2-4 days postnatal age. We used 2-amino-5-phosphonovaleric acid (AP5; 100 microM) to suppress spontaneous and stimulus-evoked polysynaptic activity. Monosynaptic EPSPs were generated by trains of 10 pulses stimuli delivered to a dorsal root at eight frequencies between 0.125 and 16 Hz. The amplitudes of the second (R2), third (R3), and the average of R8, R9, and R10 (tail) EPSPs, normalized by the first EPSP (R1), defined the shapes of synaptic depression curves. Tail responses were increasingly depressed as stimulation frequency increased but R2 and R3 exhibited relative facilitation at frequencies >1 Hz. Control experiments indicated that the depression curves were not explained by presynaptic activation failure. Lowering external Ca(2+) concentration ([Ca(2+)](o)) from 2.0 to 0.8 mM without changing [Mg(2+)](o) reduced average R1 amplitudes and R2 depression with little change in tail depression. Conversely, increasing [Ca(2+)](o) to 4.0 mM increased average R1 amplitude and R2 depression but again did not change tail depression. Increasing the bath temperature from 24 to 32 degrees C produced little change in R1 amplitudes but markedly reduced the depression of all responses at most frequencies. We developed an empirical model, based on mechanisms described in more accessible synaptic systems, that assumes: transmitter is released from a constant fraction, f, of release-ready elements in two presynaptic compartments (N and S) that are subsequently renewed by independent processes with exponential time constants (tau(N) and tau(S)); an activation-dependent facilitation of transmitter release with constant increment and fast exponential decay; and a more slowly decaying, activation-dependent augmentation of the rate of renewal (tau(N)) of N. The model gave satisfactory fits to data from all [Ca(2+)](o) conditions and implied that f and the increments of the facilitation and augmentation processes were all changed in the same direction as [Ca(2+)](o), without changing the time constants. In contrast, model fits to the 32 degrees C data implied that the process time constants all decreased by 40-45% while the presumably Ca(2+)-related weighting factors were unchanged. The model also successfully matched the normalized amplitudes of EPSPs during trains with irregular intervals.

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells.

Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.

Animal models of induced apoptotic death in the substantia nigra.

Apoptosis is a form of cell death in which genetically regulated programs intrinsic to the cell bring about its own demise. In recent years, there has been a tremendous growth of interest in apoptosis as a mechanism of disease in a wide range of human disorders including the neurodegenerative diseases, such as Parkinson's disease (PD) (1). This growth of interest has spawned an extraordinary number of recent discoveries about the molecular basis of apoptosis. It is important to emphasize, however, that much of this new knowledge has been attained in the study of relatively simple systems, such as invertebrate models or mammalian nonneural cell culture systems. Less is known about these mechanisms in neural cells, and much of what is known is based on studies of peripheral neural cells (such as sympathetic ganglia and PC 12 cells) in tissue culture. Much less is known about central neurons; in particular, we know little about the regulation of apoptotic death in central neurons in living animals. It is especially important to try to identify the mechanisms of cell death in central neurons of known phenotype, particularly those implicated in human neurodegenerative disease, such as the dopamine neurons in PD. The purpose of the models we have developed of induced apoptosis in dopamine neurons of the substantia nigra (SN) is to try to translate what is being learned about the molecular mechanisms of apoptosis in simpler systems to these neurons.

Apoptotic morphology in phenotypically defined dopaminergic neurons of the substantia nigra.

In the preceding chapter we described three paradigms by which we have studied programmed cell death in the substantia nigra (SN) of living animals: natural cell death and death induced either by developmental injury to the target striatum or by dopamine terminal destruction with the neurotoxin 6-hydroxydopamine (6-OHDA). In each of these paradigms, in order to relate experimental investigations specifically to dopamine neurons, the dopaminergic phenotype must be identified in conjunction with the demonstration of apoptotic morphology. This identification is essential, because apoptosis has been recognized in diverse neuronal populations and in glia (1).

Comparison of alternative designs for reducing complex neurons to equivalent cables.

Reduction of the morphological complexity of actual neurons into accurate, computationally efficient surrogate models is an important problem in computational neuroscience. The present work explores the use of two morphoelectrotonic transformations, somatofugal voltage attenuation (AT cables) and signal propagation delay (DL cables), as bases for construction of electrotonically equivalent cable models of neurons. In theory, the AT and DL cables should provide more accurate lumping of membrane regions that have the same transmembrane potential than the familiar equivalent cables that are based only on somatofugal electrotonic distance (LM cables). In practice, AT and DL cables indeed provided more accurate simulations of the somatic transient responses produced by fully branched neuron models than LM cables. This was the case in the presence of a somatic shunt as well as when membrane resistivity was uniform.

Developmental cell death in dopaminergic neurons of the substantia nigra of mice.

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) undergo natural cell death during development in rats. Controversy exists as to the occurrence of this phenomenon in SNpc dopaminergic neurons in the developing mouse. Herein, by using an array of morphologic techniques, we show that many SNpc neurons fulfill the criteria for apoptosis and that the number of apoptotic neurons in the SNpc vary in a time-dependent manner from postnatal day 2 to 32. These dying neurons also show evidence of DNA fragmentation, of activated caspase-3, and of cleavage of beta-actin. Some, but not all of the SNpc apoptotic neurons still express their phenotypic marker tyrosine hydroxylase, confirming their dopaminergic nature. Consistent with the importance of target-derived trophic support in modulating developmental cell death, we demonstrate that destruction of intrinsic striatal neurons by a local injection of quinolinic acid (QA) dramatically enhances the magnitude of SNpc apoptosis and results in a lower number of adult SNpc dopaminergic neurons. Strengthening the apoptotic nature of the observed SNpc developmental cell death, we demonstrate that overexpression of the anti-apoptotic protein Bcl-2 attenuates both natural and QA-induced SNpc apoptosis. The present study provides compelling evidence that developmental neuronal death with a morphology of apoptosis does occur in the SNpc of mice and that this process plays a critical role in regulating the adult number of dopaminergic neurons in the SNpc.

Elimination of the interferences by keto-opiates in the GC-MS analysis of 6-monoacetylmorphine.

A simple procedure to eliminate the interference from keto-opiates in the analysis of 6-monoacetylmorphine (6-MAM) by gas chromatography-mass spectrometry is described. The pretreatment of urine samples with sodium bisulfite followed by solid-phase extraction results in the elimination of the bisulfite addition products formed from the reaction of the bisulfite ions with the carbonyl carbon of the keto-opiates. This simple procedure results in the accurate quantitation of 6-MAM at a concentration of 4 ng/mL in the presence of keto-opiates up to 10,000 ng/mL.

Upregulation of cytosolic branched chain aminotransferase in substantia nigra following developmental striatal target injury.

We have previously shown that apoptotic cell death can be induced in substantia nigra (SN) by developmental striatal target lesion. In this model, only a portion of nigral neurons dies, so it provides a paradigm to examine not only the molecular basis of cell death, but also the cellular responses of adjacent neurons which survive. Using a differential display approach, we have found that cytosolic branched chain aminotransferase (BCATc) mRNA is upregulated in SN in this model. This upregulation is associated with an increased number of BCATc-positive neuronal profiles, demonstrated by immunostaining. BCATc-positive neurons show normal morphology and rarely contain apoptotic chromatin. We conclude that BCATc is upregulated in neurons, which are likely to survive, and plays a role in either maintenance of viability or restoration of normal function.