Matthew J. Hejna

Neurokinin-3 receptor distribution in rat and human brain: an immunohistochemical study

Autoradiographic and immunohistochemical studies have shown that the neurokinin-3 receptor is widely distributed in the rodent CNS. Expression of the neurokinin-3 receptor in human brain, however, has been debated. These conflicting findings, as well as the poor resolution of autoradiographic images, prompted us to develop a polyclonal antibody against an oligopeptide derived from the carboxy-terminus consensus sequence of both the rat and human neurokinin-3 receptor ([C]ASTTSSFISSPYTSVDEYS, amino acids 434-452 of the rat neurokinin-3 receptor). Western blot analysis of both human and rat brain tissue revealed a major band in the molecular weight range 65,000-67,000, the proposed molecular weight of the neurokinin-3 receptor based on its amino acid sequence and presumed glycosylation state. The distribution of selective high affinity neurokinin-3 receptor agonist [3H]senktide binding and neurokinin-3 receptor immunoreactivity were virtually identical in the brains of male Fischer 344 rats. The highest concentrations of neurokinin-3 receptors were observed in cortical layers IV-V; the basolateral amygdaloid nucleus; the hypothalamic paraventricular, perifornical and supraoptic nuclei; the zona incerta; and the entopeduncular and interpeduncular nuclei. [3H]senktide binding and neurokinin-3 receptor immunoreactivity were compared in homologous cortical areas of the human and rat brain. In contrast to the rat, autoradiographic analysis of normal control human brains (35-75 years) revealed a distinct and predominant superficial cortical labeling in the glia limitans and the cortical layer I. However, neurokinin-3 receptor immunoreactivity could be found not only in the superficial cortical layers, but also on pyramidal neurons and astrocytes in the neuropil and white matter. These findings suggest species differences in both the cellular and anatomical distribution of the neurokinin-3 receptor.

Local and distant histopathological effects of unilateral amyloid-β 25-35 injections into the amygdala of young F344 rats

To determine if amyloid-beta (A beta) induces tau-immunoreactivity (IR) and reactive astrocytosis in vivo, we injected A beta 25-35 (5.0 nmol) into the right amygdala of rats. At 8 days postinjection, the peptide induced tau-2 IR in neuronal cell bodies and processes ipsilaterally in the amygdala, cingulate cortex, and hippocampus. At 32 days postinjection, the intensity of tau-2 IR was greater than at 8 days in the amygdala and hippocampus, but not in the cingulate cortex. Induction of Alz-50 IR also was progressive but the morphology and distribution was different from tau-2 IR. Beaded fibers with occasional neuronal perikarya were visualized with Alz-50, and the IR was primarily observed in the ipsilateral amygdala. In addition, amygdaloid injections of A beta 25-35 induced reactive astrocytosis, particularly in the ipsilateral hippocampus at 32 days postoperatively. To our knowledge, this is the first study to show that in vivo injections of A beta 25-35 induce progressive transsynaptic cytoskeletal and astrogliotic reactions, that gradually spread from the area of injection to brain regions that have prominent efferent connections with that area. These findings also suggest a direct association between plaque and tangle formation in Alzheimers disease.

Expression of calcipressin1, an inhibitor of the phosphatase calcineurin, is altered with aging and Alzheimer's disease

Protein phosphatase 2B (calcineurin) activity has been shown to be decreased in Alzheimers disease and is a possible mechanism(s) for the hyperphosphorylation of tau and subsequent neurofibrillary tangle formation. Recently, mRNA expression of Downs syndrome Critical Region 1 gene, which encodes the protein calcipressin (an endogenous inhibitor of calcineurin), was found to be upregulated in both Downs syndrome and Alzheimers disease. Calcipressin is induced by oxidative stress and Abeta in vitro, further establishing a link in the pathology of both diseases. Using immunohistochemistry techniques, calcipressin protein expression in the pyramidal neurons of the temporal lobe was shown to increase with aging (r2=0.5658; p=0.0313), and also in moderate to severe Alzheimers disease compared to control patients (t=3.872; p=0.0017). In addition, there was a positive correlation between the total number of calcipressin-positive pyramidal neurons and the number of neurofibrillary tangles in the temporal cortex (r2= 0.5955; p=0.0249). As there was an 88% increase in nuclear calcipressin in Alzheimers disease (p=0.0001), the relationship between cellular localization of calcipressin and neurofibrillary tangle formation was investigated, which revealed a decrease in neurofibrillary tangle-bearing neurons that contain nuclear calcipressin (t=4.874; p=0.0028) and further demonstrates that the cellular regulation of calcipressin is altered in Alzheimers disease.

Oral and subcutaneous administration of the glycosaminoglycan C3 attenuates Aβ(25–35)-induced abnormal tau protein immunoreactivity in rat brain

High molecular weight glycosaminoglycans (GAG) and proteoglycans (PG) affect pathological changes of the brain in Alzheimers disease (AD). PG stimulate the processing and aggregation of amyloid-beta (Abeta), protect the protein from proteolysis, and increase the formation of neurofibrillary tangles by inducing the hyperphosphorylation of tau protein. These effects may be competitively inhibited by GAG. We have studied the effects of orally (by gavage) and subcutaneously (s.c.) administered low molecular weight heparin, C3 (4-10 oligosaccharides; MW = 2.1 kDa; USP value = 12 U/mg), on abnormal tau-2 protein immunoreactivity in the rat hippocampus following a single, unilateral intra-amygdaloid administration of Abeta(25-35). Oral administration of C3 (25 mg/kg; once daily) was initiated 3 days prior to Abeta(25-35) administration, and was continued daily for an additional 14 days. S.c. administration of C3 (2.5 mg/kg, twice daily), was started 3 days prior to, and was continued for 32 days after, Abeta(25-35) administration. Animal brains were subsequently processed for tau-2, ChAT-immunoreactivity, choline acetyltransferase (ChAT) activity and acetylcholinesterase (AChE) activity. Both oral and s.c. administration of C3 attenuated Abeta(25-35) induced appearance of tau-2-immunoreactive (IR) perikarya in the ipsilateral hippocampus (P < 0.05). Hippocampal cholinergic enzyme activity in C3 treated animals was not significantly different from control animals. The present findings suggest that C3 might be used successfully to prevent abnormal tau protein formation in chronic neurologic diseases, such as AD. Moreover, our data demonstrate that the mechanism of this effect does not appear to influence the cholinergic system of the brain.

The blood–brain barrier accessibility of a heparin-derived oligosaccharides C3

Although heparin-derived oligosaccharide(s) (HDO) have been clinically used for the management of neurological disorders, such as stroke and Alzheimers disease (AD), very little information on the mechanism of their therapeutic action is known. To test the hypothesis that HDO may pass through the blood-brain barrier (BBB) to mediate their effects, a pharmacodynamic (PD) model was developed and the presence of HDO in the cerebrospinal fluid (CSF) was used as a BBB accessibility index. Rats were treated with an ultralow molecular weight (MW) heparin fragment C3 via the intravenous or subcutaneous routes at 5-10 mg/kg. At varying periods, the plasma, CSF, and brain samples were collected, and functional anti-factor Xa activities were measured to quantitate the CSF/plasma ratios (CPR) and the brain uptake. C3 showed CPR of 1.7% and 0.8% after intravenous and subcutaneous injections, respectively. These findings were verified by intravenous administration of tritium-labeled C3 followed by detection of the radioactivity in the CSF and brain homogenates. These data suggest that ultralow MW HDO may pass through the BBB.

Low molecular weight glycosaminoglycan blockade of β-amyloid induced neuropathology

Previous studies have shown different roles for proteoglycans and glycosaminoglycans (GAGs) in Alzheimers disease (AD) neuropathology. Using a rat model of beta-amyloid induced neuropathology, we tested whether low molecular weight glycosaminoglycans (Certoparin and C6) could be useful as preventative agents and/or as a potential therapeutic treatment for AD. Chronic subcutaneous low molecular weight glycosaminoglycan injections beginning either before or after an intra-amygdaloid beta-amyloid-(25-35) injection blocked abnormal intracellular tau changes and reactive astrocytosis but did not affect beta-amyloids aggregation state. Also, low molecular weight glycosaminoglycan injections beginning 1 day prior to sacrifice did not block the effects of beta-amyloid nor did injections of a disaccharide, suggesting chronic low molecular weight glycosaminoglycan treatment is needed to block the effects of beta-amyloid. Furthermore, these data indicate that there is a molecular weight range of active low molecular weight glycosaminoglycans in this model; and supports the investigation of low molecular weight glycosaminoglycans as a preventative and/or therapeutic treatment of beta-amyloid induced neuropathology.

Age and species-dependent differences in the neurokinin B system in rat and human brain

Neurokinin B and its cognate neurokinin-3 receptor are expressed more in the forebrain than in brain stem structures but little is known about the primary function of this peptide system in the central processing of information. In general, few studies have specifically addressed age-related changes of tachykinins, notably the changes in number and/or distribution of the neurokinin B-expressing and neurokinin-3 receptor-bearing neurons. Data on functions and changes of neurokinins in physiological aging are limited and apply mainly to the substance P/neurokinin-1 receptor system. In the present study, we analyzed neurokinin B/neurokinin-3 receptor system in young (5 months) versus middle aged (15 months) and old rats (23-25 months) and also in aging human brains. For the majority of the immunohistochemically examined regions of the rat brain, there was no statistically significant change in neuronal number and size of the neurokinin B and neurokinin-3 receptor staining. In the adult human brain, there was no age-associated change of the number or size of neurokinin-B-positive neurons. However, we found a major decline in number of neurokinin-3 receptor-expressing neurons between young/middle aged (30 years to 69 years) versus old (70 years and older) adults. Interestingly, numbers of neurokinin-3 receptor-positive microglia increased whereas the neurokinin-3 receptor-positive astrocytes remained unchanged in both aging rat and human brains. Finally, in addition to assessing the morphological and quantitative changes of the neurokinin B/neurokinin-3 receptor system in the rat and human brain, we discuss functional implications of the observed interspecies differences.

Dietary supplementation with blueberry extract improves survival of transplanted dopamine neurons

Abstract The exact mechanisms contributing to poor neuronal survival in cell transplantation paradigms for Parkinsons disease (PD) are unknown. However, transplantation-induced host immune response, inflammation, and subsequent oxidative stress are likely contributors to cell death since dopamine (DA) neurons are exquisitely sensitive to oxidative damage. Multiple studies have attempted to improve cell survival by treating transplant material with antioxidant and antiinflammatory compounds, whereas far fewer studies have attempted to modify the host environment to reduce these threats. Flavonoids, phytochemicals found in fruits and vegetables, have antioxidant, antiinflammatory, and immunomodulatory properties. For example, supplementation with dietary blueberry extract (BBE) prevents oxidative stress-associated impairment of striatal motor function during aging and restores lost motor function in aged rats. We hypothesized that dietary supplementation of rodent diets with BBE would improve the survival of embryonic DA neurons transplanted into the unilaterally DA-depleted striatum. Inclusion of 2% BBE in a custom chow diet significantly increased the survival of implanted DA neurons and ameliorated rotational behavior asymmetries as compared to transplanted animals consuming a standard diet. These findings provide support for the potential of dietary phytochemicals as an easily administered and well-tolerated therapy that can be used to improve the effectiveness of DA neuron replacement.

Laterality in the histological effects of injections of amyloid-β 25-35 into the amygdala of young Fischer rats

We have observed that single amyloid-beta 25-35 (A beta) injections (5.0 nmol) into the right amygdala of rats produce progressive cytoskeletal and astrogliotic reactions not only within the amygdala, but also in distal brain regions that project to the amygdala. To determine if these effects are potentiated by bilateral injections, we injected A beta (5.0 nmol) into the left and right amygdala of young male Fischer rats. Animals were sacrificed 32 days postoperatively. Bilateral infusions of A beta induced significant neuronal shrinkage, tau-2 neuronal staining, and reactive astrocytosis within the right amygdala and/or hippocampus, compared with vehicle-treated rats. Surprisingly, the same brain regions within the left hemisphere were significantly less affected even though no differences were observed between the left and right amygdala in the size of Congored-positive A beta deposits. Unilateral injections of A beta into the left amygdala led to significant histological changes in the right amygdala and hippocampus, but not in the same brain regions within the left hemisphere. These results suggest a laterality in the histopathological effects of A beta in male Fischer rats. Identification of the cause for the lateralized effect of A beta may prove valuable for understanding the etiology of Alzheimer disease and provide possible therapeutic strategies designed to slow the progression of the disease.

β-Amyloid 25–35 and/or quinolinic acid injections into the basal forebrain of young male Fischer-344 rats: Behavioral, neurochemical and histological effects

beta-Amyloid peptides have been shown to potentiate the neurotoxic effect of excitatory amino acids in vitro. In order to determine if this occurs in vivo, four experiments were performed. We injected beta-amyloid 25-35 (beta A 25-35) and/or quinolinic acid (QA) bilaterally into the ventral pallidum/substantia innominata (VP/SI) of rats. Control rats received vehicle infusions. A high dose of QA (75.0 nmol/3 microliters) increased open field activity and impaired spatial learning in the Morris water maze, but did not affect the acquisition of a one-way conditioned avoidance response. These changes were associated with histological evidence of neurotoxicity and a reduction in amygdaloid but not frontal cortical or hippocampal choline acetyltransferase (ChAT) activity. A lower dose of QA (37.5 nmol/3 microliters) produced no behavioral effects. It reduced amygdaloid ChAT activity to a lesser extent than the higher dose (15% vs. 29-37%), and caused less histological damage. beta A 25-35 (1.0 or 8.0 nmol/3 microliters) failed to produce behavioral, histological or neurochemical signs of toxicity. Neither dose of beta A 25-35 potentiated the effects of QA (37.5 nmol) on behavior or amygdaloid ChAT activity, and did not appear to increase the histological damage caused by QA. These results suggest that in vivo beta A 25-35 is not neurotoxic and does not potentiate the neurotoxicity of QA in the VP/SI. Further, the histological effects of a high dose of beta A 25-35 (8.0 nmol/3 microliters; a cavitation containing a Congo red positive proteinaceous material) are quite distinct from those produced by a high dose of QA (75.0 nmol/3 microliters; widespread neuronal loss and gliosis).