John A. Watt

Neurotoxin-induced ER stress in mouse dopaminergic neurons involves downregulation of TRPC1 and inhibition of AKT/mTOR signaling

Individuals with Parkinsons disease (PD) experience a progressive decline in motor function as a result of selective loss of dopaminergic (DA) neurons in the substantia nigra. The mechanism(s) underlying the loss of DA neurons is not known. Here, we show that a neurotoxin that causes a disease that mimics PD upon administration to mice, because it induces the selective loss of DA neurons in the substantia nigra, alters Ca²⁺ homeostasis and induces ER stress. In a human neuroblastoma cell line, we found that endogenous store-operated Ca²⁺ entry (SOCE), which is critical for maintaining ER Ca²⁺ levels, is dependent on transient receptor potential channel 1 (TRPC1) activity. Neurotoxin treatment decreased TRPC1 expression, TRPC1 interaction with the SOCE modulator stromal interaction molecule 1 (STIM1), and Ca²⁺ entry into the cells. Overexpression of functional TRPC1 protected against neurotoxin-induced loss of SOCE, the associated decrease in ER Ca²⁺ levels, and the resultant unfolded protein response (UPR). In contrast, silencing of TRPC1 or STIM1 increased the UPR. Furthermore, Ca²⁺ entry via TRPC1 activated the AKT pathway, which has a known role in neuroprotection. Consistent with these in vitro data, Trpc1⁻/⁻ mice had an increased UPR and a reduced number of DA neurons. Brain lysates of patients with PD also showed an increased UPR and decreased TRPC1 levels. Importantly, overexpression of TRPC1 in mice restored AKT/mTOR signaling and increased DA neuron survival following neurotoxin administration. Overall, these results suggest that TRPC1 is involved in regulating Ca²⁺ homeostasis and inhibiting the UPR and thus contributes to neuronal survival.

GABAB Receptor Activation Inhibits Neuronal Excitability and Spatial Learning in the Entorhinal Cortex by Activating TREK-2 K+ Channels

The entorhinal cortex (EC) is regarded as the gateway to the hippocampus and thus is essential for learning and memory. Whereas the EC expresses a high density of GABA(B) receptors, the functions of these receptors in this region remain unexplored. Here, we examined the effects of GABA(B) receptor activation on neuronal excitability in the EC and spatial learning. Application of baclofen, a specific GABA(B) receptor agonist, inhibited significantly neuronal excitability in the EC. GABA(B) receptor-mediated inhibition in the EC was mediated via activating TREK-2, a type of two-pore domain K(+) channels, and required the functions of inhibitory G proteins and protein kinase A pathway. Depression of neuronal excitability in the EC underlies GABA(B) receptor-mediated inhibition of spatial learning as assessed by Morris water maze. Our study indicates that GABA(B) receptors exert a tight control over spatial learning by modulating neuronal excitability in the EC.

Trans‐4‐hydroxy‐2‐hexenal is a neurotoxic product of docosahexaenoic (22:6; n‐3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n‐3) acid (DHA) is elevated in the CNS in patients with Alzheimer’s disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans‐4‐hydroxy‐2‐hexenal (HHE), a six carbon analog of the n‐6 fatty acid derived trans‐4‐hydroxy‐2‐nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD50’s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration‐ and time‐dependent manners. The time‐dependent formation of HHE differed from that of F4‐neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5‐fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.

TRPC1 inhibits apoptotic cell degeneration induced by dopaminergic neurotoxin MPTP/MPP+

Disturbances in Ca(2+) homeostasis have been implicated in a variety of neuropathological conditions including Parkinsons disease (PD). However, the importance of store-operated Ca(2+) entry (SOCE) channels in PD remains to be investigated. In the present study, we have scrutinized the significance of TRPC1 in 1-methyl-4-phenyl-1,2,3,6-tetrahyrdro-pyridine (MPTP)-induced PD using C57BL/6 animal model and PC12 cell culture model. Both sub-acute and sub-chronic treatments of MPTP significantly reduced TRPC1, and tyrosine hydroxylase levels, but not TRPC3, along with increased neuronal death. Furthermore, MPTP induces mitochondrial dysfunction, which was associated with reduced mitochondrial membrane potential, decreased level of Bcl(2), Bcl-xl, and an altered Bcl-xl/Bax ratio thereby initiating apoptosis. Importantly, TRPC1 overexpression in PC12 cells showed significant protection against MPP(+) induced neuronal apoptosis, which was attributed to the restoration of cytosolic Ca(2+) and preventing loss of mitochondrial membrane potential. Silencing of TRPC1 or addition of TRPC1 channel blockers decreased mitochondrial membrane potential, whereas activation of TRPC1 restored mitochondrial membrane potential in cells overexpressing TRPC1. TRPC1 overexpression also inhibited Bax translocation to the mitochondria and thereby prevented cytochrome c release and mitochondrial-mediated apoptosis. Overall, these results provide compelling evidence for the role of TRPC1 in either onset/progression of PD and restoration of TRPC1 levels could limit neuronal degeneration in MPTP mediated PD.

Interleukin-1? immunoreactivity in identified neurons of the rat magnocellular neurosecretory system: Evidence for activity-dependent release

Interleukin‐1β has been demonstrated in neurons of the rat hypothalamus, including cells of the magnocellular neurosecretory system and tuberoinfundibular system (Lechan et al., [1990] Brain Res. 514:135–140). Despite its potential importance to regulation of neuroendocrine function, however, neither the specific cell types that express interleukin‐1β or the conditions that may result in its release have yet been described. Therefore, we utilized a combination of immunocytochemical and immunoelectron microscopic localization, in conjunction with Western blot analysis, on normonatremic, hypernatremic, and lactating rats to assess the site of synthesis and potential secretion characteristics of interleukin‐1β in the rat magnocellular neurosecretory system. Interleukin‐1β immunoreactivity was localized within both oxytocin and vasopressin neurons in the paraventricular, supraoptic, accessory and periventricular hypothalamic nuclei. Additionally, interleukin‐1β immunoreactive fibers were localized in the zona interna and zona externa of the median eminence and in the neurohypophysis. Immunoelectron microscopic analysis revealed that interleukin‐1β immunoreactivity is associated with small spherical structures, distinct from neurosecretory granules, in neurosecretory axons within the neurohypophysis. Furthermore, stimulation of heightened neurosecretory activity via chronic osmotic challenge and lactation resulted in a marked diminution in levels of interleukin‐1β immunoreactivity in the neurohypophysis with a subsequent return to normal levels after cessation of the stimuli. Western blot analysis confirmed the existence of interleukin‐1β protein in the neurohypophysis and provided further evidence for reduction in levels of IL‐1β immunoreactivity after stimulation of secretory activity. These results suggest an endogenous neuronal source of interleukin‐1β exists within the rat magnocellular neurosecretory system under normal physiological conditions. The potential for activity‐dependent release of IL‐1β and implications for the involvement of interleukin‐1β in regulation of neurosecretory activity are discussed. J. Neurosci. Res. 60:478–489, 2000

Keratin immunoreactivity in the Late Cretaceous bird Rahonavis ostromi

ABSTRACT Immunohistochemical studies, supported by additional lines of evidence, suggest that original proteinaceous components of keratin claw sheath material may be preserved in the pedal ungual phalanx associated with the primitive bird, Rahonavis ostromi, from the Late Cretaceous of Madagascar. This conclusion is based upon immunohistochemical analyses, and supported by brightfield, transmission, and scanning electron microscopy, mass spectrometry, and x-ray photoelectron spectroscopy. Although keratinous structures such as hair, nails, claws, scales and feathers have been identified in the fossil record, these identifications were based on morphological similarity rather than molecular analyses. Chemical or immunological evidence for the survival of keratin has not previously been established in fossils older than ~33,000 BP. This study demonstrates immunological staining and amino acid composition consistent with the presence of fragments of beta keratin, a protein family unique to reptiles and bird...

Acetate supplementation attenuates lipopolysaccharide-induced neuroinflammation

J. Neurochem. (2011) 117, 264–274.

Ciliary neurotrophic factor is expressed in the magnocellular neurosecretory system of the rat in vivo: Evidence for injury- and activity-induced upregulation

Although ciliary neurotrophic factor (CNTF) has been shown to promote the survival of magnocellular neurons when applied exogenously to explants of the paraventricular and supraoptic nuclei (SON) in vitro, little is known regarding its expression or regulation in the adult magnocellular neurosecretory system (MNS) following injury in vivo. Therefore, we utilized in situ hybridization and immunocytochemical analysis in conjunction with quantitative optical densitometric analysis to identify the cellular source of CNTF and examine the temporal pattern of its expression, following unilateral transection of the hypothalamo-neurohypophysial tract in the adult rat. In intact rats, CNTF immunoreactivity (CNTF-ir) was predominantly localized within identified astrocytes within the ventral glial limitans subjacent to the SON. Quantitative optical densitometric analysis of CNTF-ir levels in the axotomized SON demonstrated that the proportional area of CNTF-ir was significantly elevated between 3 and 30 days following injury. A significant but more limited increase was also observed in the non-injured contralateral SON. In situ hybridization confirmed the expression and upregulation of CNTF in the axotomized SON. These results demonstrate the expression of CNTF in the adult rodent MNS in vivo and provide evidence that levels of CNTF are upregulated in response to both direct injury, and heightened metabolic activity, within the lesioned and sprouting SON, respectively.

The multifaceted responses of primary human astrocytes and brain microvascular endothelial cells to the Lyme disease spirochete, Borrelia burgdorferi.

The vector-borne pathogen, Borrelia burgdorferi, causes a multi-system disorder including neurological complications. These neurological disorders, collectively termed neuroborreliosis, can occur in up to 15% of untreated patients. The neurological symptoms are probably a result of a glial-driven, host inflammatory response to the bacterium. However, the specific contributions of individual glial and other support cell types to the pathogenesis of neuroborreliosis are relatively unexplored. The goal of this project was to characterize specific astrocyte and endothelial cell responses to B. burgdorferi. Primary human astrocytes and primary HBMEC (human brain microvascular endothelial cells) were incubated with B. burgdorferi over a 72-h period and the transcriptional responses to the bacterium were analyzed by real-time PCR arrays. There was a robust increase in several surveyed chemokine and related genes, including IL (interleukin)-8, for both primary astrocytes and HBMEC. Array results were confirmed with individual sets of PCR primers. The production of specific chemokines by both astrocytes and HBMEC in response to B. burgdorferi, including IL-8, CXCL-1, and CXCL-10, were confirmed by ELISA. These results demonstrate that primary astrocytes and HBMEC respond to virulent B. burgdorferi by producing a number of chemokines. These data suggest that infiltrating phagocytic cells, particularly neutrophils, attracted by chemokines expressed at the BBB (blood–brain barrier) may be important contributors to the early inflammatory events associated with neuroborreliosis.

Compensatory sprouting of uninjured magnocellular neurosecretory axons in the rat neural lobe following unilateral hypothalamic lesion

Axonal sprouting of intact neurons of the magnocellular neurosecretory system was investigated using a unilateral hypothalamic knife cut of the hypothalamoneurohypophysial tract to partially denervate the rat neural lobe (NL). Densitometric, morphometric, ultrastructural, and metabolic measures were utilized to demonstrate the compensatory response to denervation in this system. Densitometric analysis revealed a transient reduction in the intensity of vasopressin staining in the NL at 10 days postsurgery (PS) with a subsequent recovery by 20 days PS. There was a comparable initial reduction in the cross-sectional area of the NL followed by a more gradual recovery to normal by 90 days PS. Ultrastructural investigation revealed a reduction in total axon number in the NL at 10 days PS similar to the declines in vasopressin immunoreactivity and size of the NL. A subsequent partial recovery of axon number occurred, paralleling the return to normal NL size between 30 and 90 days PS. Hypertrophy of both somata and cell nuclei of magnocellular neurons in the supraoptic and paraventricular hypothalamic nuclei contralateral to the lesion was also apparent during this period. Daily measurements of urine osmolality revealed an initial transient hypoosmolality followed by a chronic hyperosmolality which persisted throughout the 90 day postsurgical period. There was a concomitant chronic decrease in both daily drinking and urine excretion volumes which began immediately following surgery. These results suggest that intact, contralateral magnocellular vasopressinergic efferents undergo compensatory sprouting as a result of partial denervation of the NL in the absence of a functional deficit in vasopressin.