Diana I. Lurie

Lead exposure during development results in increased neurofilament phosphorylation, neuritic beading, and temporal processing deficits within the murine auditory brainstem

Low‐level lead (Pb) exposure is a risk factor for learning disabilities, attention deficit hyperactivity disorder (ADHD), and other neurological dysfunction. It is not known how Pb produces these behavioral deficits, but low‐level exposure during development is associated with auditory temporal processing deficits in an avian model, while hearing thresholds remain normal. Similar auditory processing deficits are found in children with learning disabilities and ADHD. To identify cellular changes underlying this functional deficit, Pb‐induced alterations of neurons and glia within the mammalian auditory brainstem nuclei were quantified in control and Pb‐exposed mice at postnatal day 21 by using immunohistochemistry, Western blotting, and 2D gel electrophoresis. Pb‐treated mice were exposed to either 0.1 mM (low) or 2 mM (high) Pb acetate throughout gestation and through 21 days postnatally. Pb exposure results in little change in glial proteins such as glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), or F4/80 as determined by Western blot analysis and immunohistochemistry. In contrast, Pb exposure alters neuronal structural proteins by inducing increased phosphorylation of both the medium (NFM) and high‐weight (NFH) forms of neurofilament within auditory brainstem nuclei. Axons immunolabeled for neurofilament protein show neuritic beading following Pb exposure both in vivo and in vitro, suggesting that Pb exposure also impairs axonal transport. Functional assessment shows no significant loss of peripheral function, but does reveal impairments in brainstem conduction time and temporal processing within the brainstem. These results provide evidence that Pb exposure during development alters axonal structure and function within brainstem auditory nuclei. J. Comp. Neurol. 506:1003–1017, 2008.

Lack of the Protein Tyrosine Phosphatase SHP-1 Results in Decreased Numbers of Glia Within the Motheaten (me/me) Mouse Brain

Mice that are homozygous for the autosomal recessive motheaten allele (me/me) lack the protein tyrosine phosphatase SHP‐1. Loss of SHP‐1 leads to many hematopoietic abnormalities, as well as defects such as infertility and low body weight. However, little is known regarding the role SHP‐1 plays in the development of the central nervous system (CNS). To define the role of SHP‐1 in CNS development and differentiation, we examined the brains of me/me mice at various times after birth for neuronal and glial abnormalities. Although the brains of me/me mice are slightly smaller than age‐matched wild‐type littermates, both me/me and wild‐type brains are similar in weight, possess an intact blood‐brain barrier, and have largely normal neuronal architecture. Significantly, the current study reveals that me/me brain shows decreases in the number of glial fibriallary acidic protein (GFAP)+ astrocytes and F480+ microglia compared with wild‐type mice. In addition, decreased immunostaining for the myelin‐synthesizing enzyme CNPase was observed in me/me mice, confirming the loss of myelin in these animals, as reported (Massa et al. [2000] Glia 29:376–385). It is particularly significant that there is a decreased number of immunolabeled glia of all subtypes and that this deficit in glial number is not restricted to a particular class of glia. This suggests that SHP‐1 is necessary for the normal differentiation and distribution of astrocytes, microglia, and oligendrocytes within the murine CNS. J. Comp. Neurol. 441:118–133, 2001.

Chronic low-level lead exposure affects the monoaminergic system in the mouse superior olivary complex.

Low‐level lead (Pb) exposure is associated with behavioral and cognitive dysfunction, but it is not clear how Pb produces these behavioral changes. Pb has been shown to alter auditory temporal processing in both humans and animals. Auditory temporal processing occurs in the superior olivary complex (SOC) in the brainstem, where it is an important component in sound detection in noisy environments and in selective auditory attention. The SOC receives a serotonergic innervation from the dorsal raphe, and serotonin has been implicated in auditory temporal processing within the brainstem and inferior colliculus. Because Pb exposure modulates auditory temporal processing, the serotonergic system is a potential target for Pb. The current study was undertaken to determine whether developmental Pb exposure preferentially changes the serotonergic system within the SOC. Pb‐treated mice were exposed to no Pb, very low Pb (0.01 mM), or low Pb (0.1 mM) throughout gestation and through 21 days postnatally. Brainstem sections from control and Pb‐exposed mice were immunostained for the vesicular monoamine transporter 2 (VMAT2), serotonin (5‐HT), and dopamine‐β‐hydroxylase (DβH; a marker for norepinephrine) in order to elucidate the effect of Pb on monoaminergic input into the SOC. Sections were also immunolabeled with antibodies to vesicular glutamate transporter 1 (VGLUT1), vesicular γ‐aminobutyric acid (GABA) transporter (VGAT), and vesicular acetylcholine transporter (VAChT) to determine whether Pb exposure alters the glutaminergic, GABAergic, or cholinergic systems. Pb exposure caused a significant decrease in VMAT2, 5‐HT, and DβH expression, whereas VGLUT1, VGAT, and VAChT showed no change. These results provide evidence that Pb exposure during development alters normal monoaminergic expression in the auditory brainstem. J. Comp. Neurol. 513:542–558, 2009.

Lipopolysaccharide‐activated SHP‐1‐deficient motheaten microglia release increased nitric oxide, TNF‐α, and IL‐1β

Accumulating evidence suggests a deleterious role for activated microglia in facilitating neuronal death by producing neurocytotoxic substances during injury, infection, or neurodegenerative diseases. After cochlear ablation, abnormal microglial activation accompanied by increased neuronal loss within the auditory brainstem occurs in motheaten (me/me) mice deficient in the protein tyrosine phosphatase SHP‐1. To determine whether abnormally activated microglia contribute to neuronal death in me/me mice, primary microglial cultures from me/me and wild‐type mouse cortices were stimulated by the bacterial endotoxin lipopolysaccharide (LPS) to evaluate the secretion of the neurotoxic mediators nitric oxide (NO), tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β). Me/me microglia release significantly greater amounts of all three mediators compared with wild‐type microglia. However, the increased release of these compounds in microglia lacking SHP‐1 does not appear to occur through activation of extracellular signal‐regulated kinase (ERK), p38 kinase subgroups of mitogen‐activated protein (MAP) kinases, or increases in NF‐κB‐inducing kinase (NIK). These results suggest that abnormal microglial activation and release of neurotoxic compounds may potentiate neuronal death in deafferented cells and can thus potentiate neurodegeneration in the me/me brainstem. Our data also indicate that SHP‐1 is engaged in signaling pathways in LPS‐activated microglia, but not through regulation of the ERK and p38 MAP kinases.

Development of Cat‐301 immunoreactivity in auditory brainstem nuclei of the gerbil

The developing brainstem auditory system has been studied in detail by using anatomical and physiological techniques. However, it is not known whether immature auditory neurons exhibit different molecular characteristics than those of physiologically mature neurons. To address this issue, we examined the distribution of Cat‐301 immunoreactivity in the developing auditory brainstem of gerbils. Cat‐301 is a monoclonal antibody that recognizes a 680‐kD chondroitin sulfate proteoglycan similar to aggrecan, a high‐molecular‐weight chondroitin sulfate proteoglycan found in cartilage. In the central nervous system, Cat‐301 immunoreactivity is localized to the extrasynaptic surface of neurons. It has been hypothesized by Hockfield and co‐workers (Hockfield et al. [1990a]Cold Spring Harbor Symp. Quart. Biol. 55:504–514) that the Cat‐301 proteoglycan is a molecular marker indicating that a neuron has acquired mature neuronal properties.

Focal cerebral ischemia upregulates SHP-1 in reactive astrocytes in juvenile mice.

The role of the tyrosine phosphatase SHP-1 in the hematopoietic system has been well studied; however, its role in the central nervous system (CNS) response to injury is not well understood. Previous studies in our laboratory have demonstrated increased immunoreactivity for SHP-1 in a subset of reactive astrocytes that do not appear to enter the cell cycle following deafferentation of the chicken auditory brainstem. In order to determine whether mammalian astrocytes also upregulate SHP-1 immunoreactivity following CNS injury, a mouse model of focal cerebral ischemia was utilized to study SHP-1 expression. The brains of 3-week-old mice were analyzed at four time points following permanent middle cerebral artery occlusion (MCAO): 1, 3, 7, and 14 days. Our results demonstrate consistent infarct volumes within surgical groups, and infarct volumes decrease as a function of time from 1 day (maximum infarct volume) to 14 days (minimum infarct volume) post-MCAO. In addition, SHP-1 protein levels are upregulated following cerebral ischemia and this increase peaks at 7 days post-MCAO. Analysis of confocal images further reveals that immunoreactivity for SHP-1 occurs predominantly in GFAP+ reactive astrocytes, although a small percentage of F4-80+ microglia are also double labeled for SHP-1 at early times post-MCAO. These SHP-1+ reactive astrocytes do not appear to enter the cell cycle (as defined by PCNA immunoreactivity), confirming our previous studies in the avian auditory brainstem. These results suggest that SHP-1 plays an important role in the regulation of glial activation and proliferation in the ischemic CNS.

Afferent Deprivation Elicits a Transcriptional Response Associated with Neuronal Survival after a Critical Period in the Mouse Cochlear Nucleus

The mechanisms underlying enhanced plasticity of synaptic connections and susceptibilities to manipulations of afferent activity in developing sensory systems are not well understood. One example is the rapid and dramatic neuron death that occurs after removal of afferent input to the cochlear nucleus (CN) of young mammals and birds. The molecular basis of this critical period of neuronal vulnerability and the transition to survival independent of afferent input remains to be defined. Here we used microarray analyses, real-time reverse transcription PCR, and immunohistochemistry of the mouse CN to show that deafferentation results in strikingly different sets of regulated genes in vulnerable [postnatal day (P) 7] and invulnerable (P21) CN. An unexpectedly large set of immune-related genes was induced by afferent deprivation after the critical period, which corresponded with glial proliferation over the same time frame. Apoptotic gene expression was not highly regulated in the vulnerable CN after afferent deprivation but, surprisingly, did increase after deafferentation at P21, when all neurons ultimately survive. Pharmacological activity blockade in the eighth nerve mimicked afferent deprivation for only a subset of the afferent deprivation regulated genes, indicating the presence of an additional factor not dependent on action potential-mediated signaling that is also responsible for transcriptional changes. Overall, our results suggest that the cell death machinery during this critical period is mainly constitutive, whereas after the critical period neuronal survival could be actively promoted by both constitutive and induced gene expression.

Decreased Expression of the Voltage-Dependent Anion Channel in Differentiated PC-12 and SH-SY5Y Cells Following Low-Level Pb Exposure

Lead (Pb) has been shown to disrupt cellular energy metabolism, which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage-dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5 and 10microM Pb on VDAC expression in vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time reverse transcription-polymerase chain reaction demonstrated a significant decrease in messenger RNA levels for the VDAC1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5 and 10microM Pb reduces VDAC transcription and expression and is associated with reduced cellular ATP levels.

Chronic low-level Pb exposure during development decreases the expression of the voltage dependent anion channel in auditory neurons of the brainstem

Lead (Pb) exposure is a risk factor for neurological dysfunction. How Pb produces these behavioral deficits is unknown, but Pb exposure during development is associated with auditory temporal processing deficits in both humans and animals. Pb disrupts cellular energy metabolism and efficient energy production is crucial for auditory neurons to maintain high rates of synaptic activity. The voltage-dependent anion channel (VDAC) is involved in the regulation of mitochondrial physiology and is a critical component in controlling mitochondrial energy production. We have previously demonstrated that VDAC is an in vitro target for Pb, therefore, VDAC may represent a potential target for Pb in the auditory system. In order to determine whether Pb alters VDAC expression in central auditory neurons, CBA/CaJ mice (n=3-5/group) were exposed to 0.01mM, or 0.1mM Pb acetate during development via drinking water. At P21, immunohistochemistry reveals a significant decrease for VDAC in neurons of the Medial Nucleus of the Trapezoid Body. Western blot analysis confirms that Pb results in a significant decrease for VDAC. Decreases in VDAC expression could lead to an upregulation of other cellular energy producing systems as a compensatory mechanism, and a Pb-induced increase in brain type creatine kinase is observed in auditory regions of the brainstem. In addition, comparative proteomic analysis shows that several proteins of the glycolytic pathway, the phosphocreatine circuit, and oxidative phosphorylation are also upregulated in response to developmental Pb exposure. Thus, Pb-induced decreases in VDAC could have a significant effect on the function of auditory neurons.

Motheaten (me/me) mice deficient in SHP-1 are less susceptible to focal cerebral ischemia

We have demonstrated previously that the protein tyrosine phosphatase SHP‐1 seems to play a role in glial development and is upregulated in non‐dividing astrocytes after injury. The present study examines the effect of loss of SHP‐1 on the CNS response to permanent focal ischemia. SHP‐1 deficient (me/me) mice and wild‐type littermates received a permanent middle cerebral artery occlusion (MCAO). At 1, 3, and 7 days after MCAO, infarct volume, neuronal survival and cell death, gliosis, and inflammatory cytokine levels were quantified. SHP‐1 deficient me/me mice display smaller infarct volumes at 7 days post‐MCAO, increased neuronal survival within the ischemic penumbra, and decreased numbers of cleaved caspase 3+ cells within the ischemic core compared with wild‐type mice. In addition, me/me mice exhibit increases in GFAP+ reactive astrocytes, F4‐80+ microglia, and a concomitant increase in the level of interleukin 12 (IL‐12) over baseline compared with wild‐type. Taken together, these results demonstrate that loss of SHP‐1 results in greater healing of the infarct due to less apoptosis and more neuronal survival in the ischemic core and suggests that pharmacologic inactivation of SHP‐1 may have potential therapeutic value in limiting CNS degeneration after ischemic stroke.