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Intranasal insulin administration raises central nervous system (CNS) insulin levels in humans and acutely facilitates verbal memory in patients with Alzheimer's disease (AD), an effect that may differ by APOE genotype. The purpose of this study was to examine the cognitive dose response curves for intranasal insulin administration, and determine whether the effects of insulin differ between participants with (epsilon4+) and without (epsilon4-) the APOE- epsilon4 allele. On separate mornings, 33 memory-impaired adults with AD or amnestic mild cognitive impairment and 59 normal adults each underwent five intranasal treatment conditions consisting of insulin (10, 20, 40, or 60 IU) or placebo. Cognition was tested 15-minutes post-treatment, and blood was acquired at baseline and 45-minutes post-treatment. Plasma insulin and glucose levels were unaffected by treatment. Insulin administration facilitated recall on two measures of verbal memory in memory-impaired epsilon4- adults, with performance generally peaking at 20 IU. In contrast, memory-impaired epsilon4+ subjects demonstrated a relative decline in verbal memory. Insulin also differentially modulated plasma amyloid-beta for memory-impaired subjects and normal controls, effects that again differed by APOE genotype. These findings suggest that groups with different genetic risks for AD may show differential dose-response curves following intranasal insulin administration.

We determined the nervous system targeting of interferon-beta1b (IFN-beta1b), a 20 kDa protein used to treat the relapsing-remitting form of multiple sclerosis, following intranasal administration in anesthetized, adult cynomolgus monkeys. Five animals received an intranasal bolus of [(125)I]-labeled IFN-beta1b, applied bilaterally to the upper nasal passages. Serial blood samples were collected for 45 min, after which the animals were euthanized by transcardial perfusion-fixation. High resolution phosphor imaging of tissue sections and gamma counting of microdissected tissue were used to obtain the distribution and concentration profiles of [(125)I]-IFN-beta1b in central and peripheral tissues. Intranasal administration resulted in rapid, widespread targeting of nervous tissue. The olfactory bulbs and trigeminal nerve exhibited [(125)I]-IFN-beta1b levels significantly greater than in peripheral organs and at least one order of magnitude higher than any other nervous tissue area sampled. The basal ganglia exhibited highest [(125)I]-IFN-beta1b levels among CNS regions other than the olfactory bulbs. Preferential IFN-beta1b distribution to the primate basal ganglia is a new finding of possible clinical importance. Our study suggests both IFN-beta and IFN-alpha, which share the same receptor, may be bound with relatively high affinity in these structures, possibly offering new insight into a neurovegetative syndrome induced by IFN-alpha therapy and suspected to involve altered dopamine neurotransmission in the basal ganglia. Most importantly, our results suggest intranasally applied macromolecules may bypass the blood-brain barrier and rapidly enter the primate CNS along olfactory- and trigeminal-associated extracellular pathways, as shown previously in the rat. This is the first study to finely detail the central distribution of a labeled protein after intranasal administration in non-human primates.

This study is aimed to evaluate the brain distribution of transforming growth factor-beta1 (TGF-beta1) following intranasal administration and the subsequent biological effects of TGF-beta1. Adult rats were given recombinant human TGF-beta1 (rhTGF-beta1) or vehicle solution intranasally. TGF-beta1 concentrations were significantly raised in several brain regions and the trigeminal nerve following intranasal delivery. The elevation appeared within 30 min and was sustained for at least 6 h, reaching its greatest level at 60 min. A concentration gradient in the central nervous system (CNS) regions was produced during the first 2 h after intranasal administration, with the OB presenting a significantly higher concentration than any other CNS regions. The nasally administered TGF-beta1 subsequently regulated gene expressions of its two receptors (TGF-beta receptor types I and II) in vivo, but did not affect mRNA level of TGF-beta1 itself. Our results suggest that TGF-beta1 can be transported into the CNS via the olfactory and trigeminal pathways, and may consequently exert its biological effects by regulating gene expressions of its receptors. Intranasal administration of neurotrophic factors may offer a potential strategy for treating some CNS disorders.

Cumulative evidence has indicated a critical role of poly(ADP-ribose) polymerase-1 activation in ischemic brain damage. Poly(ADP-ribose) glycohydrolase (PARG) is a key enzyme in poly(ADP-ribose) catabolism. Our previous studies showed that PARG inhibitors, gallotannin (GT) and nobotanin B, can profoundly decrease oxidative cell death in vitro. Here, we tested the hypothesis that intranasal delivery of GT can decrease ischemic brain damage by inhibiting PARG. Intranasal delivery of 25 mg / kg GT within 5 hours after a 2-hour focal brain ischemia markedly decreased the infarct formation and neurological deficits of rats. The GT administration also increased poly(ADP-ribose) in the ischemic brains, suggesting that GT acts as a PARG inhibitor in vivo. Furthermore, the GT treatment abolished nuclear translocation of apoptosis-inducing factor (AIF) in the ischemic brains, suggesting that prevention of AIF translocation may contribute to the protective effects of GT. In contrast, intravenous injection of GT, at 2 hours after ischemic onset, did not reduce ischemic brain damage. Collectively, our findings suggest that PARG inhibition can significantly decrease ischemic brain injury, possibly by blocking AIF translocation. This study also highlights distinct merits of intranasal drug delivery for treating CNS diseases.

Several studies have demonstrated aberrations in the Electron Transport Complexes (ETC) and Krebs (TCA) cycle in Alzheimer's disease (AD) brain. Optimal activity of these key metabolic pathways depends on several redox active centers and metabolites including heme, coenzyme Q, iron-sulfur, vitamins, minerals, and micronutrients. Disturbed heme metabolism leads to increased aberrations in the ETC (loss of complex IV), dimerization of APP, free radical production, markers of oxidative damage, and ultimately cell death all of which represent key cytopathologies in AD. The mechanism of mitochondrial dysfunction in AD is controversial. The observations that Abeta is found both in the cells and in the mitochondria and that Abeta binds with heme may provide clues to this mechanism. Mitochondrial Abeta may interfere with key metabolites or metabolic pathways in a manner that overwhelms the mitochondrial mechanisms of repair. Identifying the molecular mechanism for how Abeta interferes with mitochondria and that explains the established key cytopathologies in AD may also suggest molecular targets for therapeutic interventions. Below we review recent studies describing the possible role of Abeta in altered energy production through heme metabolism. We further discuss how protecting mitochondria could confer resistance to oxidative and environmental insults. Therapies targeted at protecting mitochondria may improve the clinical outcome of AD patients.

Alzheimer's disease (AD) is the major cause of dementia, accounting for 50% to 70% of the late-onset patients, with 17 to 20 million affected. It is characterized by neurofibrillary tangles, neuronal loss, and amyloid plaques in tissues of the cortex, hippocampus, and amygdala. Apoptosis or programmed cell death appears in the progression of AD. In this study, we investigated the gene expression of 14 apoptotic genes (E2F1, p21/WAF, ICE-LAP3, Fas Antigen, CPP-32, GADD153, ICE-beta, c-Fos, c-Jun, Bax-alpha, Bcl-2, Bcl-(x)L, BAK, and p53) in 5 normal and 6 AD human hippocampal tissues, using reverse transcription-polymerase chain reaction. Our results show an upregulation of gene expression in AD patients for c-Fos and BAK. ICE-beta, c-Jun, Bax-alpha, Bcl-x(L), p53, and GADD153 were found to be upregulated in some AD samples but were not detected or downregulated in other AD or normal samples. No gene expression was found for E2F1 , p21/WAF, ICE-LAP3, Fas Antigen, CPP32, or Bcl-2. These results indicate significant increases in c-Fos , c-Jun, and Bak; therefore, we suggest that these genes may be critical in the apoptotic cascades of AD.

Intranasal drug administration is a noninvasive method of bypassing the blood-brain barrier (BBB) to deliver neurotrophins and other therapeutic agents to the brain and spinal cord. This method allows drugs that do not cross the BBB to be delivered to the central nervous system (CNS) and eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways. Intranasal delivery occurs by an extracellular route and does not require that drugs bind to any receptor or undergo axonal transport. Intranasal delivery also targets the nasal associated lymphatic tissues (NALT) and deep cervical lymph nodes. In addition, intranasally administered therapeutics are observed at high levels in the blood vessel walls and perivascular spaces of the cerebrovasculature. Using this intranasal method in animal models, researchers have successfully reduced stroke damage, reversed Alzheimer's neurodegeneration, reduced anxiety, improved memory, stimulated cerebral neurogenesis, and treated brain tumors. In humans, intranasal insulin has been shown to improve memory in normal adults and patients with Alzheimer's disease. Intranasal delivery strategies that can be employed to treat and prevent NeuroAIDS include: (1) target antiretrovirals to reach HIV that harbors in the CNS; (2) target therapeutics to protect neurons in the CNS; (3) modulate the neuroimmune function of moncyte/macrophages by targeting the lymphatics, perivascular spaces of the cerebrovasculature, and the CNS; and (4) improve memory and cognitive function by targeting therapeutics to the CNS.

Intranasal (IN) delivery of HIV-1 Tat in aging mice was investigated as a possible model for HIV-1 infection in the brain. After IN administration, the distribution of [(125)I]-labeled Tat in the brains of Swiss Webster mice was evaluated by autoradiography and gamma counting. [(125)I]-labeled Tat was detected at the highest concentrations in the olfactory bulb, cervical nodes, and trigeminal nerve tract. In another experiment, APPSw transgenic mice were used to model chronic Tat exposure. The mice were treated intranasally with 6 mug Tat (n = 4) or vehicle (n = 4) three times per week for 4 weeks. Total RNA was isolated from the frontal cortex, and differential gene expression analysis was performed using gene microarrays. Gene ontology profiles indicated innate immunity, inflammatory and apoptotic responses. Five genes of interest in the Tat-treated mice that were significantly elevated in the microarrays were validated by RT-PCR. One gene, the Toll-like receptor 9 (Tlr9), has previously been shown to activate signaling cascades leading to innate immunity and enhanced HIV-1 gene expression. A second gene, Fas, plays a key role in neuroinflammation. Two cysteine-rich cytokines associated with chemotaxis were elevated: MCP-1 (Ccl2), which is chemotactic for monocytes, and Ccl17 (TARC), which is chemotactic for lymphocytes. Finally, the gene sestrin was significantly elevated and has been associated with oxidative stress, in particular amyloid beta-induced oxidative stress. This IN Tat model of neuroinflammation may be useful to study HIV-1-induced neurodegeneration.

We assessed the presence and characteristics of the intracellular pathogen Chlamydophila (Chlamydia) pneumoniae in brain-tissue samples from 25 patients with late-onset Alzheimer's disease (AD) and 27 non-AD control individuals. 20/27 AD patients, but only 3/27 controls, were PCR-positive in multiple assays targetting the Cpn1046 and Cpn0695 genes. Culture of the organism from brain-tissue homogenate from one AD patient, and assessment of various chlamydial transcripts in RNA preparations from several patients, demonstrated that the organisms were viable and metabolically active in those samples. Immunohistochemical analyses showed that astrocytes, microglia, and neurons all served as host cells for C. pneumoniae in the AD brain, and that infected cells were found in close proximity to both neuritic senile plaques and neurofibrillary tangles in the AD brain. These observations confirm and significantly extend our earlier study suggesting that this unusual pathogen may play a role in the neuropathogenesis characteristic of AD.

Raising insulin acutely in the periphery and in brain improves verbal memory. Intranasal insulin administration, which raises insulin acutely in the CNS without raising plasma insulin levels, provides an opportunity to determine whether these effects are mediated by central insulin or peripheral processes. Based on prior research with intravenous insulin, we predicted that the treatment response would differ between subjects with (epsilon4+) and without (epsilon4-) the APOE-epsilon4 allele. On separate mornings, 26 memory-impaired subjects (13 with early Alzheimer's disease and 13 with amnestic mild cognitive impairment) and 35 normal controls each underwent three intranasal treatment conditions consisting of saline (placebo) or insulin (20 or 40 IU). Cognition was tested 15 min post-treatment, and blood was acquired at baseline and 45 min after treatment. Intranasal insulin treatment did not change plasma insulin or glucose levels. Insulin treatment facilitated recall on two measures of verbal memory in memory-impaired epsilon4- adults. These effects were stronger for memory-impaired epsilon4- subjects than for memory-impaired epsilon4+ subjects and normal adults. Unexpectedly, memory-impaired epsilon4+ subjects showed poorer recall following insulin administration on one test of memory. These findings suggest that intranasal insulin administration may have therapeutic benefit without the risk of peripheral hypoglycemia and provide further evidence for apolipoprotein E (APOE) related differences in insulin metabolism.

Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-b (P < 0.01) and 26% less heme-a (P = 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in heme-a/heme-b ratio (P < 0.001). Moreover, the strong Pearson correlation between heme-a and heme-b measured in control individuals (r(2) = 0.66, P < 0.002, n = 11) was abolished in AD subjects (r(2) = 0.076, P = 0.39, n = 12). The level of ferrochelatase (which makes heme-b in the mitochondrial matrix) in AD subjects was 4.2 times (P < 0.04) that in nondemented controls, suggesting up-regulated heme synthesis. To look for a possible connection between these observations and established mechanisms in AD pathology, we examined possible interactions between amyloid beta (A beta) and heme. A beta((1-40)) and A beta((1-42)) induced a redshift of 15-20 nm in the spectrum of heme-b and heme-a, suggesting that heme binds A beta, likely to one or more of the histidine residues. Lastly, in a tissue culture model, we found that clioquinol, a metal chelator in clinical trials for AD therapy, decreased intracellular heme. In light of these observations, we have proposed a model of AD pathobiology in which intracellular A beta complexes with free heme, thereby decreasing its bioavailability (e.g., heme-a) and resulting in functional heme deficiency. The model integrates disparate observations, including A beta, mitochondrial dysfunction, cholesterol, and the proposed efficacy of clioquinol.

Intranasal (i.n.) administration of IFN beta-1b was examined as a route for targeted delivery to the rat central nervous system (CNS). Intranasal administration resulted in significant delivery throughout the CNS and cervical lymph nodes with low delivery to peripheral organs. At similar blood levels, intravenous (i.v.) administration of IFN beta-1b yielded 88-98% lower CNS levels and 100-1650% greater peripheral organ levels compared to intranasal. Autoradiography confirmed much greater delivery to the CNS with intranasal administration. Intranasally administered IFN beta-1b reached the brain intact and produced tyrosine phosphorylation of IFN receptor in the CNS. Intranasal administration offers a non-invasive method of drug delivery for multiple sclerosis (MS) that bypasses the blood-brain barrier (BBB) and directly targets the CNS and lymph nodes.

We investigated the CNS delivery of insulin-like growth factor-I (IGF-I), a 7.65 kDa protein neurotrophic factor, following intranasal administration and the possible pathways and mechanisms underlying transport from the nasal passages to the CNS. Anesthetized adult male Sprague-Dawley rats were given [125I]-IGF-I intranasally or intravenously and then killed by perfusion-fixation within 30 min. Other animals were killed following cisternal puncture and withdrawal of cerebrospinal fluid (CSF) or intranasal administration of unlabeled IGF-I or vehicle. Both gamma counting of microdissected tissue and high resolution phosphor imaging of tissue sections showed that the tissue concentrations and distribution following intranasal administration were consistent with two routes of rapid entry into the CNS: one associated with the peripheral olfactory system connecting the nasal passages with the olfactory bulbs and rostral brain regions (e.g. anterior olfactory nucleus and frontal cortex) and the other associated with the peripheral trigeminal system connecting the nasal passages with brainstem and spinal cord regions. Intranasal administration of [125I]-IGF-I also targeted the deep cervical lymph nodes, consistent with their possible role in lymphatic drainage of both the nasal passages and the CNS. Cisternal CSF did not contain [125I]-IGF-I following intranasal administration. Intravenous [125I]-IGF-I resulted in blood and peripheral tissue exposure similar to that seen following intranasal administration but CNS concentrations were significantly lower. Finally, delivery of IGF-I into the CNS activated IGF-I signaling pathways, confirming some portion of the IGF-I that reached CNS target sites was functionally intact. The results suggest intranasally delivered IGF-I can bypass the blood-brain barrier via olfactory- and trigeminal-associated extracellular pathways to rapidly elicit biological effects at multiple sites within the brain and spinal cord.

Intracerebroventricular injection of insulin-like growth factor (IGF)-I has been shown to protect against stroke in rats. This method of delivery is not practical in human beings, as it requires an operation with risk of infection and other complications. Intranasal (i.n.) delivery offers a noninvasive method of bypassing the blood-brain barrier to deliver IGF-I to the brain. This study delineates the window of opportunity for treatment of focal cerebral ischemic damage using i.n. IGF-I after middle cerebral artery occlusion (MCAO). Rats were allowed to survive 7 days after 2 hours of MCAO. Infarct volume, apoptosis after 7 days, and neurologic deficit scores from the postural reflex and adhesive tape tests assessing motor-sensory and somatosensory functions, respectively, at 1 to 7 days were used to evaluate the efficacy of i.n. IGF-I (150 microg) administered at different times after MCAO. I.n. IGF-I significantly reduced infarct volume by 54%; and 39%; versus control when administered at 2 or 4 hours, respectively, after the onset of MCAO (P < .05) and improved motor-sensory and somatosensory functions (P < .05) when administered 2 hours after the onset of MCAO. In addition, treatment with i.n. IGF-I at 2, 4, or 6 hours after MCAO decreased apoptotic cell counts by more than 90%; in the hemisphere ipsilateral to the occlusion. I.n. IGF-I is a promising treatment for stroke with a therapeutic window of opportunity for up to 6 hours after the onset of ischemia. This noninvasive method provides a simpler, safer, and potentially more cost-effective method of delivery than other methods currently in use.

Although head imaging studies are frequently used in the work-up of dementia, published criteria for the clinical diagnosis of Alzheimer's disease (AD) do not require them. Since our brain bank contains cases in which physicians had specifically diagnosed AD without using a head imaging study, we thought it of interest to investigate the accuracy of their clinical diagnoses. We retrospectively reviewed 911 consecutive dementia cases for those clinically diagnosed as either AD or senile dementia (SD). Twenty-one were identified in which head imaging studies had not been used, each diagnosed as AD or SD by a different physician. In only three had the physician reported a reason why a study was not done. In all 21 cases the primary neuropathological cause of the dementia was AD. Neuropathology in addition to AD was also noted, including cortical Lewy bodies in three, infarcts on gross examination in three, multiple microscopic infarcts in four, and multiple cerebral metastases in one. Acknowledging a number of study limitations, it is remarkable that the judgment of the physicians was correct regarding AD in all 21 cases. It is questionable if a head CT or MRI scan at time of diagnosis would have benefited any of the patients.

Oxidative stress has been implicated as a contributing factor to neurodegeneration in Alzheimer's disease. An endogenous, low molecular weight (LMW) inhibitor from Alzheimer's brain inactivates the human brain muscarinic acetylcholine receptor (mAChR). The inhibitor prevents agonist and antagonist binding to the mAChR as assessed by radioligand binding studies. The LMW endogenous inhibitor, which has components with molecular weights between 100 and 1000 Da, requires dissolved oxygen and glutathione. Prevention of inactivation of the mAChR with peroxidase suggests that the LMW endogenous inhibitor generates peroxide. Heme, previously shown to be present in the LMW endogenous inhibitor, also inactivates the mAChR in the presence of peroxide. Free radical damage to the muscarinic receptor by the endogenous inhibitor can be prevented through the use of naturally occurring antioxidants including bilirubin, biliverdin, carnosol, myricetin and quericetin. In addition, pyrophosphate, imidodiphosphate, bisphosphonates and related compounds also protect the muscarinic receptor from free radical damage. Inactivation of the mAChR by the LMW endogenous inhibitor is likely to be a factor in the continual decline of Alzheimer's patients, even those taking acetylcholinesterase inhibitors. Natural antioxidants and pyrophosphate analogs may improve the effectiveness of acetylcholinesterase inhibitors and prove useful in the treatment and prevention of Alzheimer's disease since the muscarinic acetylcholine receptor is required for memory, and decreased cholinergic function is a critical deficit in Alzheimer's disease.

Insulin-like growth factor-I (IGF-I) has been proposed as a treatment for stroke. However, it does not efficiently cross the blood-brain barrier (BBB). Intracerebroventricular injection of IGF-I has been shown to offer protection against cerebral ischemic damage in rats although this invasive method of administration may not be practical in humans. Non-invasive intranasal (IN) delivery of IGF-I to the brain is a promising alternative. We have assessed the therapeutic effect of IN IGF-I in rats following middle cerebral artery occlusion (MCAO). Treatment was initiated 10 min after the onset of MCAO and then again 24 and 48 h later. Intranasal dosing of 75 microg IGF-1 (225 microg total IGF-I over 48 h) significantly reduced corrected infarct volumes by 60% vs. control (P<0.01) and hemispheric swelling by 45.6% vs. control (P<0.05). Neurologic function, assessed by the postural reflex, flexor response and adhesive tape tests, was also improved by IN IGF-I as compared to control. Our study indicates IN delivery of IGF-1 holds significant promise as a non-invasive and efficacious method of bypassing the BBB for the treatment of stroke.

Background: Insulin-like growth factor-I (IGF-I) has been shown to protect against stroke in rats when administered intracerebroventricularly. However, this invasive method of administration is not practical for the large number of individuals who require treatment for stroke. Intranasal (IN) delivery offers a noninvasive method of bypassing the blood-brain barrier (BBB) to deliver IGF-I and other neurotrophic factors to the brain. Here, we demonstrate for the first time the therapeutic benefit of IN IGF-1 in rats following middle cerebral artery occlusion (MCAO)., Methods: A blinded, vehicle-controlled study of IN IGF-I was performed using the intraluminal suture occlusion model. Rats were randomly divided into vehicle-control, 37.5 and 150 microg IGF-I-treated groups. Treatments occurred at 10 min after onset of 2 h of MCAO, and then 24 and 48 h later. Four neurologic behavioral tests were performed 4, 24, 48 and 72 h after the onset of MCAO. Corrected infarct volumes were evaluated 72 h after the onset of MCAO., Results: Treatment with the 150 microg IGF-I significantly reduced the infarct volume by 63% vs. control (p=0.004), and improved all the neurologic deficit tests of motor, sensory, reflex and vestibulomotor functions (p<0.01). However, the 37.5 microg dose of IGF-I was ineffective., Conclusion: While IGF-I does not cross the BBB efficiently, it can be delivered to the brain directly from the nasal cavity following IN administration, bypassing the BBB. IN IGF-I markedly reduced infarct volume and improved neurologic function following focal cerebral ischemia. This noninvasive, simple and cost-effective method is a potential treatment for stroke.

Neurotrophic factors are proteins with considerable potential in the treatment of central nervous system (CNS) diseases and traumatic injuries. However, a significant challenge to their clinical use is the difficulty associated with delivering these proteins to the CNS. Neurotrophic factors are hydrophilic, typically basic, monomeric or dimeric proteins, mostly in the size range of 5 to 30 kDa. Neurotrophic factors potently support the development, growth and survival of neurons, eliciting biological effects at concentrations in the nanomolar to femtomolar range. They are not orally bioavailable and the blood-brain and blood-cerebrospinal fluid barriers severely limit their ability to enter into and act on sites in the CNS following parenteral systemic routes of administration. Most neurotrophic factors have short in vivo half-lives and poor pharmacokinetic profiles. Their access to the CNS is restricted by rapid enzymatic inactivation, multiple clearance processes, potential immunogenicity and sequestration by binding proteins and other components of the blood and peripheral tissues. The development of targeted drug delivery strategies for neurotrophic factors will probably determine their clinical effectiveness for CNS conditions. Achieving significant CNS target site concentrations while limiting systemic exposure and distribution to peripheral sites of action will lessen unwanted pleiotropic effects and toxicity. Local introduction of neurotrophic factors into the CNS intraparenchymally by direct injection/infusion or by implantation of delivery vectors such as polymer matrices or genetically modified cells yields the highest degree of targeting, but is limited by diffusion restrictions and invasiveness. Delivery of neurotrophic factors into the cerebrospinal fluid (CSF) following intracerebroventricular or intrathecal administration is less invasive and allows access to a much wider area of the CNS through CSF circulation pathways. However, diffusional and cellular barriers to penetration into surrounding CNS tissue and significant clearance of CSF into the venous and lymphatic circulation are also limiting. Unconventional delivery strategies such as intranasal administration may offer some degree of CNS targeting with minimal invasiveness. This review presents a summary of the neurotrophic factors and their indications for CNS disorders, their physicochemical characteristics and the different approaches that have been attempted or suggested for their delivery to the CNS. Future directions for further research such as the potential for CNS disease treatment utilising combinations of neurotrophic factors, displacement strategies, small molecule mimetics, chimaeric molecules and gene therapy are also discussed.

In a retrospective cliniconeuropathological study, we reviewed all the cases received in our dementia brain bank during a 4-year period to determine if all patients with severe substantia nigra (SN) degeneration and SN Lewy bodies (LBs) exhibited prominent signs of parkinsonism and were treated for parkinsonism during the disease course. The SN of 426 cases were graded for microscopic degeneration using a semiquantitative five-tiered scale, with grade 0 indicating normal and grade 4 the most severe degeneration. Twenty-nine cases with grade 3 (16) or grade 4 (13) SN degeneration with SN LBs and clinical records were identified. Ten had been treated for parkinsonism (6 grade 3, 4 grade 4) and 19 had not. Whereas most of the patients had exhibited signs of end-stage parkinsonism during their last year, 1 grade 3 and 2 grade 4 patients apparently never exhibited prominent signs of parkinsonism during the course of their dementia. No clear neuropathological differences were noted between these patients that did not have prominent signs and a control group of six patients with clinical Parkinson's disease with dementia (parkinsonism onset at least 1 year before dementia onset). We conclude that in patients with dementia there is an inconsistent relationship between the expression of clinical parkinsonism during life and severe SN degeneration with LBs identified at necropsy.

Objectives: To identify patients with pure hippocampal sclerosis (HS) as a cause of dementia, to determine whether they have had histories of hypotension or hypoxia, and to compare the clinical features of patients with pure HS with a control group of AD patients without HS., Methods: In a retrospective study, the authors reviewed all 1771 cases received in their dementia brain bank from 1978 through 1996 to identify those patients with pure HS, defined as severe degeneration and gliosis of the CA1 sector and subiculum of the hippocampal formation in the absence of other significant dementing disease such as Alzheimer's changes. The control group included all patients received during the same period with severe AD without HS, infarcts, or other dementing disease., Results: Seven pure HS cases (0.4%) were identified. None had any episodes of syncope, hypotension, or hypoxia reported in association with dementia onset. Six had memory loss as the primary presenting symptom, and all became progressively demented. Forty-five AD patients without HS were identified for the control group. There were no clear clinical differences between the two groups with regard to sex, age at onset, risk factors for vascular disease, symptoms of cerebrovascular disease, treatment with tranquilizing medications, treatment for depression, or nursing home placement. There was a tendency for heart disease to be more prevalent and the duration of illness to be shorter in the patients with pure HS., Conclusions: Pure hippocampal sclerosis (HS) occurred in only 0.4% of our dementia patients. Clinically, the seven patients with pure HS were similar to our AD control group. Further research is needed to determine the causes of HS and why HS appears to mimic AD.

Loss of memory and cholinergic transmission are associated with both Alzheimer's disease (AD) and marijuana use. The human brain muscarinic acetylcholine receptor (mAChR), which is involved in memory function and is inhibited by arachidonic acid, is also inhibited by anandamides. Two agonists of the cannabinoid receptor derived from arachidonic acid, anandamide (AEA) and R-methanandamide, inhibit ligand binding to the mAChR. Binding of the mAChR antagonist [3H]quinuclidinyl benzilate ([3H]QNB) is inhibited up to 89% by AEA (half-maximal inhibition at 50 microM). Binding of the more polar antagonist [N-methyl-3H]scopolamine ([3H]NMS) is inhibited by AEA up to 76% (half-maximal inhibition at 44 microM). R-methanandamide inhibits more than 90% of both [3H]QNB binding (I50 = 34 microM) and [3H]NMS binding (I50 = 15 microM) to the mAChR. Both AEA and R-methanandamide stimulate mAChR binding of the agonist [3H]oxotremorine-M at low concentrations (25-75 microM), but significantly inhibit agonist binding at higher concentrations (I50 = 150 microM). The cannabinoid antagonist SR141716A did not alter AEA or R-methanandamide inhibition of [3H]NMS binding to the mAChR, even at concentrations as high as 1 microM. Further, the cannabinoid agonist WIN 55212-2 does not alter antagonist binding to the mAChR. This demonstrates that mAChR inhibition by the anandamides is not mediated by the cannabinoid receptor. Since AEA and R-methanandamide are structurally similar to arachidonic acid, they may interact with the mAChR in a similar manner to inhibit receptor function.

In a retrospective clinicopathological study, we examined the substantia nigra (SN) of 48 dementia patients with SN Lewy bodies (LBs) to determine if the severity of degeneration correlated with either the occurrence of signs of parkinsonism at dementia presentation or with the frequency of treatment for parkinsonism during the disease course. The SN specimens were graded for microscopic degeneration using a semi-quantitative five-tiered scale. Whereas no correlation was found between the grade of degeneration and occurrence of signs at presentation (r = -0.16, p = 0.18), with 16 of 38 patients having had signs reported, a more severe grade was statistically correlated with an increased frequency of treatment during the course (r = 0.41, p = 0.004), with ten of 41 patients having been treated for parkinsonism. Contrary to our expectations, we found that fewer than half of the patients with the two most severe grades of degeneration presented with signs of parkinsonism or were ever treated for parkinsonism.

Arachidonic acid (AA) inhibits the binding of [3H]quinclidinyl benzilate ([3H]QNB) to the human brain muscarinic cholinergic receptor (mAChR). AA inhibits at lower concentrations in the absence of glutathione (I50 = 15 microM) than in the presence of glutathione (I50 = 42 microM). Inhibition of mAChR binding shows specificity for AA and is reduced with loss of one or more double bonds or with either a decrease or increase in the length of the fatty acid chain. Metabolism of AA by the lipoxygenase, epoxygenase, or fatty acid cyclooxygenase pathways is not required for the inhibitory activity of AA on mAChR binding. Inhibition of [3H]QNB binding by AA is reversible. While decreasing Bmax, AA increased the apparent KD for [3H]QNB and for the more polar antagonist [3H]NMS. In addition, AA inhibits binding of the agonist [3H]oxotremorine-M (I50 = 60 microM) and is the first mediator of mAChR action to be shown to reversibly inhibit mAChR binding. The feedback inhibition of the mAChR by AA may serve a homeostatic function similar to the reuptake and hydrolysis of acetylcholine following cholinergic nerve transmission.

An endogenous inhibitor (< 3500 Da) of antagonist binding to the muscarinic acetylcholine receptor (mAChR) has been reported to be elevated 3-fold in Alzheimer's disease (AD) brain. This endogenous inhibitor was found to require the presence of reducing agents such as reduced glutathione (GSH) for optimal activity. In the presence of GSH, the inhibitor was shown to generate thiyl radicals which irreversibly inhibited the mAChR. We now report that the inhibitor contains free heme, a well-established source of oxidative stress capable of generating free radicals and causing neurotoxicity. While FeSO4, microperoxidase and hemin all inhibited antagonist binding to the mAChR, only hemin shared the inhibitor's requirement for GSH. Both the free radical scavengers Trolox and Mn2+, and the metal chelator, EDTA, blocked the activity of the endogenous AD inhibitor and of hemin. Heme oxygenase-1 (HO-1) markedly reduced the activity of both the endogenous AD inhibitor and hemin, indicating that the endogenous inhibitor contains heme. Mass spectrometric analysis confirmed the presence of free heme and heme fragments in fractions of the endogenous AD inhibitor. The antioxidants estrogen, vitamin E and vitamin C all protected the mAChR from irreversible inhibition by the endogenous inhibitor or hemin. These antioxidants may function to protect the integrity of the mAChR in vivo and may have therapeutic potential in AD where free heme could be a source of oxidative stress.

Metavanadate, orthovanadate, and pervanadate all inhibited [3H]QNB antagonist binding to the human brain muscarinic acetylcholine receptor (mAChR) in the presence of glutathione, with the order of decreasing potency and the concentration required for 50% inhibition (I[50]) being: pervanadate (95 microM) > orthovanadate (132 microM) > metavanadate (452 microM). Omission of glutathione decreased the inhibition of the vanadium compounds 2-6-fold. Preincubating the vanadium compounds with the mAChR in the presence of glutathione at 37 degrees for 1 h markedly decreased the I(50) values as follows: pervanadate (13 microM) > orthovanadate (46 microM) > metavanadate (118 microM). Inhibition by the vanadium compounds was blocked by EDTA, Mn2+, and Trolox, a water-soluble vitamin E analog. Vanadium use in treating diabetes is discussed regarding its inhibition of mAChR function.

Objectives: To compare, in a retrospective clinicopathological study, the presentation features of patients with dementia and cortical Lewy bodies (Lewy body dementia) with those of patients with Alzheimer's disease., Methods: From a population of 426 cases from the dementia brain bank, 39 cases of Lewy body dementia and 61 cases of Alzheimer's disease with presentation details were identified., Results: The Lewy body dementia group had significantly more frequent hallucinations (23% v 3%, P = 0.006) and signs of parkinsonism (41% v 5%, P < 0.0001) than the Alzheimer's disease group. The Lewy body dementia group also had a greater proportion of men (62% v 34%, P = 0.013)., Conclusion: Hallucinations and signs of parkinsonism help distinguish Lewy body dementia from Alzheimer's disease at presentation. These indicators may not be very sensitive, because they were reported for less than half of the patients with Lewy body dementia.

An endogenous inhibitor (< 3,500 Da) of antagonist binding to the muscarinic acetylcholine receptor has been extracted from Alzheimer's disease (AD) brain with trifluoracetic acid. Oxidized glutathione, (GSSG) has also been found to inhibit antagonist binding to the receptor. However, in its reduced form, glutathione (GSH) like other reducing agents, markedly enhances the inhibitory effect of both GSSG and the endogenous AD inhibitor. EDTA and the free radical scavengers Mn(2+) and Trolox, a vitamin E analog, block the action of the endogenous AD inhibitor but not of GSSG in the presence of GSH. Further, while GSSG inhibition is reversible, the action of the endogenous AD inhibitor is irreversible, consistent with a free radical mechanism. The enhancement of endogenous AD inhibitor activity by GSH suggested that GSH may be involved in formation of the free radical generated by the inhibitor. The glutathione thiyl radical is shown to inhibit antagonist binding to the receptor and is, therefore, a good candidate for the free radical formed by the endogenous AD inhibitor. The ability of Trolox to block the reduction in muscarinic receptor binding caused by the endogenous AD inhibitor is encouraging and suggests that free radical scavengers, such as vitamin E, may have a potential therapeutic role in AD by protecting the integrity of the muscarinic receptor.

Following intranasal administration to rats, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) concentrated in the olfactory nerve and glomerular layers of the olfactory bulb resulting in a mean olfactory bulb concentration of 140 nM. A negligible amount of label was detected in the olfactory bulb following intravenous administration of WGA-HRP or intranasal administration of unconjugated HRP. This is the first quantitative assessment of intraneuronal transport of a protein into the brain using the olfactory route.

The NINCDS-ADRDA criteria for the clinical diagnosis of probable Alzheimer disease (AD) state that "gait disturbances at the onset or very early in the course of the illness make the diagnosis of Alzheimer disease uncertain or unlikely," yet there have been few studies documenting the validity of the statement. We therefore reviewed all cases of pure autopsy-proven AD in the Ramsey Brain Bank to determine how frequently an abnormal gait was noted at time of presentation. Any reported gait disturbance was considered an abnormal gait. Only cases for which medical records were available documenting the patient's presentation for dementia were included. Cases were excluded if any other pathology was present that may have contributed to the patient's dementia or to a gait disorder, if neuroleptic medication had been used, or if there was a preexisting gait disorder. Clinical dementia severity at time of presentation was graded as mild, moderate, or severe per DSM-IIIR criteria. Of the 95 cases that met study criteria, none of the 36 patients with mild dementia were reported to have had an abnormal gait. Sixteen percent of the patients with moderate and 32% with severe dementia had gait abnormalities reported. This study confirms the statement regarding gait in the NINCDS-ADRDA criteria.

This is the first report of the presence of free D-amino acids in lumbar and ventricular human cerebrospinal fluid (CSF) of individuals with Alzheimer disease (AD) compared with CSF of normal control subjects and with individuals affected by multiple sclerosis, as an unrelated neurologic disorder. Free D-amino acids are present at significantly higher levels in AD CSF than normal CSF, whereas in the CSF of patients affected by multiple sclerosis, D-amino acids occurs at the same level as in the normal controls. The total D-amino acid content in ventricular CSF was 1.48 times higher in the AD than controls (26.4 vs 17.9 nmol/mL, p = 0.025). The total D-amino acid content was 1.43 times higher in AD lumbar CSF than controls (1.89 vs 1.32 nmol/mL, p = 0.001). D-Aspartate in particular was 2.74 times higher in AD ventricular CSF compared to normal ventricular CSF (3.34 vs 1.22 nmol/mL, p = 0.029). In lumbar CSF, D-aspartate was 1.5 times higher in AD than controls (0.054 vs 0.036 nmol/mL, p = 0.041). Previously we reported that D-amino acids are elevated in AD brain proteins associated with neurofibrillary tangles compared to normal brain proteins (D'Aniello et al., 1992c; Fisher et al., 1992a,b). Thus, the D-amino acids present in CSF may originate from degradation of brain proteins.

New-onset epileptic seizures occur in patients with Alzheimer's disease (AD), but the nature and underlying reasons for these seizures are unclear. We identified new-onset, nonsymptomatic seizures in 77 (17%) of 446 patients with uncomplicated, definite AD on autopsy. Among these seizure patients, 69 had generalized tonic-clonic seizures, and 55 had less than three total seizures. The seizure patients had a younger age of dementia onset than did the remaining AD patients; however, at seizure onset, they averaged 6.8 years into their AD and had advanced dementia. When further compared to 77 AD controls, matched for age of onset and duration, the seizure patients did not differ on medical illnesses, amount of medications, and degree of focal neuropathology. We conclude that a few unprovoked generalized seizures frequently occur late in the course of AD, and that AD patients with a younger age of dementia onset are particularly susceptible to seizures.

Chronic Creutzfeldt-Jakob disease, presenting in later life, may be difficult to distinguish from other dementing illnesses. Patients with this disease may lack the characteristic myoclonus and electroencephalographic complexes. We report four patients with a slowly progressive dementia who were misdiagnosed during life and had a spongiform encephalopathy at autopsy. All four patients had early extrapyramidal rigidity, primitive reflexes, and other neurologic signs. The presence of these neurologic findings early in the course of a dementia suggests that clinicians should consider chronic Creutzfeldt-Jakob disease, evaluate the patient with electroencephalographic studies, and emphasize universal tissue precautions.

The 100,000 x g supernatant fraction of human brain contains endogenous inhibitors of antagonist binding to the muscarinic receptor. Significantly greater inhibition was observed with Alzheimer's than non-demented control supernatant fractions. Low molecular weight inhibitor was separated from larger inhibitor species by membrane dialysis (3,500 dalton cut-off). The activity of low molecular weight inhibitor was greatly increased by sulfhydryl reducing agents. While the low molecular weight inhibitor was stable to heat, acid and base for short time periods (< 20 min), it was inactivated by acid hydrolysis (50% loss after 16 h, 100% loss after 96 h). The low molecular weight inhibitor activity is elevated approximately three-fold in Alzheimer's brain. The low molecular weight inhibitor from Alzheimer's brain was found to be a non-competitive inhibitor. This is the first report of endogenous inhibitors in human brain of ligand binding to the muscarinic receptor and of increased inhibitor activity in Alzheimer's disease.

During a study evaluating GM1 ganglioside as a possible treatment for Alzheimer's disease, two patients suffered immune responses that appeared to be limited to localized inflammation at the sites of the intramuscular GM1 injections. We determined that one patient's anti-GM1 IgM antibody titer rose from 1:400 to 1:3200 and her anti-GM1 IgG titer from < 1:50 to 1:400,000 during the immune response. The second patient's titer rose from < 1:50 to 1:3200 IgM and from 1:3200 to 1:400,000 IgG. These findings document that patients may experience acute rises in their anti-GM1 antibody levels in response to GM1 and that such rises may not necessarily cause significant acute clinical neuronal injury.

Alzheimer's disease (AD) has been proposed to be an inflammatory disorder. In a recent study, markedly elevated levels of the anti-inflammatory cytokine transforming growth factor beta (TGF-beta) in the serum and cerebrospinal fluid of patients with advanced AD suggested a potential predictive value of this cytokine in patients with AD. In the present prospective study, we tested the hypothesis that the levels of TGF-beta in serum would be increased in patients with AD and could thereby serve as a diagnostic marker. We found that serum TGF-beta levels but not proinflammatory cytokine levels were significantly (P < 0.05) elevated in patients with AD (n = 22) in comparison with the levels in their healthy spousal controls. Also, serum TGF-beta levels were positively correlated (r = 0.45; P < 0.05) with disease severity. Nevertheless, the elevation in serum TGF-beta levels in patients with Ad was modest, and considerable overlap with the control values suggests that the diagnostic usefulness of this cytokine for AD is limited.

Alzheimer's disease (AD) has been hypothesized to be an inflammatory condition. We hypothesized that anti-inflammatory cytokines, such as transforming growth factor beta (TGF-beta), counteract the inflammatory process. In the present study, we found that TGF-beta levels were elevated in both cerebrospinal fluid and serum samples obtained from AD patients < 6 h after death. Serum TGF-beta levels were also markedly elevated before death. These results suggest that elevated TGF-beta levels in AD may represent a protective host response to immunologically mediated neuronal injury.

This paper describes a new type of ELISA plate in which the reaction wells have been coated with silica gel. ELISA using these prototype silica-ELISA plates is markedly more sensitive for glycolipid antigens in lipid mixtures than ELISA using polystyrene plates without silica. Silica-ELISA plates also improve the analysis of certain protein and carbohydrate antigens. This technology may be of considerable benefit in the analysis of minor lipids and other antigens from human brain, cerebrospinal fluid or blood.

The clinical recognition of Pick's disease depends on its differentiation from Alzheimer's disease (AD). To identify distinguishing clinical features, we reviewed the clinical records of 21 patients with pathologically confirmed Pick's disease and matched them by sex, age of onset, and duration of dementia with 42 patients having pathologically confirmed AD. In the absence of temporal or frontal lobar atrophy on CTs, all the Pick patients and none of the AD patients had three of five clinical features: presenile onset (before age 65), an initial personality change, hyperorality, disinhibition, and roaming behavior. In addition, the Pick patients had a tendency toward reiterative and other speech disturbances. These findings suggest that Pick patients are potentially distinguishable from AD patients on the basis of clinical manifestations.

Objective: To investigate the accuracy of cranial computerized tomography (CT) scans in distinguishing patients with Alzheimer's disease from those with other dementing conditions., Design: Retrospective clinicopathological correlation with pre-mortem CT scans., Setting: Urban and rural hospitals and nursing homes in the Upper Midwest., Patients: All 507 patients had clinical dementia diagnosed as Alzheimer's disease during life and the subsequent referral of their brains to a dementia brain bank. Of these, 375 patients had had CT scans as part of the diagnostic work-up for dementia., Main Outcome Measures: The presence of neuropathological evidence of Alzheimer's disease and the specific findings on CT scans., Results: Of the 375 patients evaluated with a CT, 28% were misdiagnosed (lacked neuropathological evidence of Alzheimer's disease); of the 132 patients evaluated without a CT scan, only 18% were misdiagnosed (P less than 0.05). The degree of atrophy and other CT findings were similar in the correctly diagnosed and misdiagnosed groups except for increased ventricular size in the correctly diagnosed patients (P less than 0.05)., Conclusion: Although CT scans do not usually contribute to the recognition of Alzheimer's disease, the presence of ventricular enlargement may help distinguish Alzheimer's disease from other dementias.

We investigated potential risk factors for Alzheimer's disease (AD) in a clinicopathologic study of 407 patients with definite AD, 100 non-Alzheimer dementia patients, and 50 normal subjects. The AD patients had more first-degree relatives with dementia than the non-AD dementia group (odds ratio of 1.85, 95% confidence interval of 1.07-3.20) or the normal elderly (odds ratio of 3.60, 95% confidence interval of 1.50-8.64) but did not have significantly more head injuries, medical and psychiatric illnesses, or relatives with Down's syndrome. The AD patients with a family history of dementia had their dementia at a later age than those without an affected relative. These findings indicate a familial risk for AD that is greater than for other dementing illnesses and has age-related penetrance. This study does not support other putative risk factors for AD such as head trauma and familial Down's syndrome.

Despite the introduction of formal clinical criteria for Alzheimer disease (AD), the clinical diagnosis of AD remains one of exclusion of other dementias. To determine the accuracy of a clinical diagnosis of AD as made by practicing physicians, we reviewed the clinicopathologic records of a dementia brain bank and summarized the literature. Of 650 demented patients diagnosed during life as having AD, at autopsy 505 (78%) had AD with or without other neuropathologic conditions; only 390 (60%) of these had AD as the only neuropathologic condition. Of the remaining 145 (22%) patients with no neuropathologic evidence of AD, 39 had the nigrostriatal changes of Parkinson disease (PD), 25 had nonspecific degenerations, 15 had Pick disease, 14 had multiple infarcts, and 11 lacked any neuropathologic abnormality. Although the overall clinical accuracy for AD was lower than that summarized from the literature, clinical accuracy improved significantly between 1986 and 1990. In our broad sample of practitioners, accuracy of clinical diagnosis of AD may be improving, but continues to be hampered by difficulty in distinguishing the dementia of AD from certain dementing conditions and from AD mixed with other neuropathologic conditions.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo post-translational modification by racemization to D-aspartate, or by isomerization to the L-isoaspartyl form in which the peptide chain links through the beta carboxyl group of the residue. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a significantly greater number of these modified aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo posttranslational modification by racemization to D-aspartate. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a greater number of these racemized D-aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

We obtained season-of-birth data in 727 autopsy-confirmed cases of Alzheimer's Disease (AD) and compared these data with expected general population birth rates. There were no significant differences between quarterly birth rates in the AD group and expected quarterly birth rates. Edward's test for cyclical trends did not establish a peak period of birth in the AD sample. No significant differences between observed and expected quarterly birth rates were found when data were analyzed with regard to either family history of dementia or to gender. Edward's test for peak quarter was significant for AD females, however, with the peak period occurring early in the first quarter. These negative findings between observed and expected quarterly birth rates, based on the large number of autopsy-confirmed AD cases in this study, suggest that a season-of-birth effect in AD is highly unlikely.

In the absence of pathognomonic clinical features, the clinical diagnosis of Alzheimer's disease (AD) remains one of exclusion of other dementias. We investigated the clinical diagnoses among 394 neuropathologically confirmed AD cases in a dementia brain bank. Most patients were correctly diagnosed as AD (348 or 88%). Among the misdiagnosed patients, AD was mistaken for a primary depressive disorder in 14, multi-infarct dementia in 13, Parkinson's disease in nine, and alcoholic dementia in four. The number of misdiagnosed AD patients did not differ between physician specialties but was greater among AD patients with agitation, depression, paranoia, or delusions. This retrospective study suggests that the diagnostic sensitivity for AD is high among a cross-section of practicing physicians and that an important factor in mistaking AD for another illness is unfamiliarity with the potential psychiatric symptoms of AD.