Olfactory Dysfunction and Alzheimer's Disease: A Review.

 Alzheimer's disease is the most common cause of dementia, and it is one of the leading causes of death globally. Identification and validation of biomarkers that herald the onset and progression of Alzheimer's disease is of paramount importance for early reliable diagnosis and effective pharmacological therapy commencement. A substantial body of evidence has emerged demonstrating that olfactory dysfunction is a preclinical symptom of neurodegenerative diseases including Alzheimer's disease. While a correlation between olfactory dysfunction and Alzheimer's disease onset and progression in humans exists, the mechanism underlying this relationship remains unknown. The aim of this article is to review the current state of knowledge regarding the range of potential factors that may contribute to the development of Alzheimer's disease-related olfactory dysfunction. This review predominantly focuses on genetic mutations associated with Alzheimer's disease including amyloid-ß protein precursor, presenilin 1 and 2, and apolipoprotein E mutations, that may (in varying ways) drive the cellular events that lead to and sustain olfactory dysfunction.

Immunohistochemical phenotype of sensory neurons associated with sympathetic plexuses in the trigeminal ganglia of adult nerve growth factor transgenic mice.

Following peripheral nerve injury, postganglionic sympathetic axons sprout into the affected sensory ganglia and form perineuronal sympathetic plexuses with somata of sensory neurons. This sympathosensory coupling contributes to the onset and persistence of injury-induced chronic pain. We have documented the presence of similar sympathetic plexuses in the trigeminal ganglia of adult mice that ectopically overexpress nerve growth factor (NGF), in the absence of nerve injury. In this study, we sought to further define the phenotype(s) of these trigeminal sensory neurons having sympathetic plexuses in our transgenic mice. Using quantitative immunofluorescence staining analyses, we show that the invading sympathetic axons specifically target sensory somata immunopositive for several biomarkers: NGF high-affinity receptor tyrosine kinase A (trkA), calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NFH), and P2X purinoceptor 3 (P2X3). Based on these phenotypic characteristics, the majority of the sensory somata surrounded by sympathetic plexuses are likely to be NGF-responsive nociceptors (i.e., trkA expressing) that are peptidergic (i.e., CGRP expressing), myelinated (i.e., NFH expressing), and ATP sensitive (i.e., P2X3 expressing). Our data also show that very few sympathetic plexuses surround sensory somata expressing other nociceptive (pain) biomarkers, including substance P and acid-sensing ion channel 3. No sympathetic plexuses are associated with sensory somata that display isolectin B4 binding. Though the cellular mechanisms that trigger the formation of sympathetic plexus (with and without nerve injury) remain unknown, our new observations yield an unexpected specificity with which invading sympathetic axons appear to target a precise subtype of nociceptors. This selectivity likely contributes to pain development and maintenance associated with sympathosensory coupling.

Serum proteomics as a strategy to identify novel biomarkers of neurologic recovery after cardiac arrest: a feasibility study.

BACKGROUND: Serum biomarkers may play a role in prognostication after cardiac arrest. This study was designed to assess the feasibility of using two-dimensional gel electrophoresis (2D-GE) coupled with mass spectrometry (MS) as a proteomic strategy to identify novel biomarkers that may predict neurological recovery. METHODS: Adult comatose survivors of ventricular fibrillation or pulseless ventricular tachycardia were considered eligible. Blood was collected and serum separated within 6 h of hospital admission and then at 24 h afterwards. Neurological outcome was assessed at 3 months with the Cerebral Performance Category (CPC) score. Serum was assessed with 2D-GE with and without prior depletion of high abundance proteins. Protein differences between patients with good (CPC 1,2) vs. poor (CPC 3-5) neurological recovery were subsequently identified with MS. RESULTS: From August 2010 to June 2014, 11 patients meeting eligibility criteria were recruited, from which serum was available from 9 (5 with good neurological outcome). On non-depleted serum, only high abundance acute phase proteins such as haptoglobin, cell-free hemoglobin, albumin, and amyloid were detected in both patients with good and poor neurological recovery. Following depletion of high abundance proteins, proteins identified by MS in both patient populations were the acute phase reactants c-reactive protein and retinol binding protein-4. Proteins uniquely identified in the serum of patients with poor neurological recovery included 14-3-3 (epsilon and zeta isoforms) and muskelin. CONCLUSIONS: Two-D-GE coupled with MS is a feasible strategy to facilitate the identification of novel predictive biomarkers. The presence of muskelin and 14-3-3 in the serum of patients with poor neurological prognosis warrants further investigation.

Myenteric expression of nerve growth factor and the p75 neurotrophin receptor regulate axonal remodeling as a consequence of colonic inflammation in mice.

Nerve growth factor (NGF) levels increase in response to inflammation of the mammalian colon. The precise cellular sources of colonic NGF synthesis, however, remain elusive. Using lines of transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the NGF promoter, we found a subpopulation of adendritic EGFP(+) neurons in the myenteric plexus. These colonic EGFP(+) neurons display positive immunostaining for calretinin but not nitric oxide synthase 1 (NOS1) two biomarkers of mouse myenteric neurons. A loss of NGF expression in null mutant postnatal mice does not affect the survival of these EGFP(+) neurons. Induction of colonic inflammation confirms local increases in NGF mRNA/protein levels, which coincide with heightened detection of EGFP by myenteric neurons. Though NOS1(+) myenteric neurons display positive immunostaining for trkA (the receptor required for NGF binding/signaling), transgenic overexpression of NGF by smooth muscle cells in the colon does not alter the survival, somal size, or axonal density of trkA-expressing NOS1(+) myenteric neurons. Mice lacking functional p75NTR (the second receptor required for NGF binding) exhibit significantly less axonal damage among NOS1(+) myenteric neurons, in response to chemically induced colonic inflammation. Likewise, trkA-expressing sympathetic axons that innervate the myenteric ganglia display less damage in the absence of p75NTR. These data are the first to implicate calretinin(+) myenteric neurons as a source of NGF in the murine colon, and that in response to colonic inflammation, increases in NGF can exaggerate damage of intrinsic NOS1(+) axons and extrinsic sympathetic axons that co-express trkA and p75NTR.

Neuronal degeneration associated with sympathosensory plexuses in the trigeminal ganglia of aged mice that overexpress nerve growth factor.

Aberrant sympathetic sprouting is seen in the uninjured trigeminal ganglia of transgenic mice that ectopically express nerve growth factor under the control of the glial fibrillary acidic protein promoter. These sympathetic axons form perineuronal plexuses around a subset of sensory somata in 2- to 3-month-old transgenic mice. Here, we show that aged transgenic mice (i.e., 11-14 and 16-18 months old) have dystrophic sympathetic plexuses (i.e., increased densities of swollen axons), and that satellite glial cells, specifically those in contact with dystrophic plexuses in the aged mice display strong immunostaining for tumor necrosis factor alpha. The colocalization of dystrophic plexuses and reactive satellite glial cells in the aged mice coincides with degenerative features in the enveloped sensory somata. Collectively, these novel results show that, with advancing age, sympathetic plexuses undergo dystrophic changes that heighten satellite glial cell reactivity and that together these cellular events coincide with neuronal degeneration.

Reactive astrocytes associated with plaques in TgCRND8 mouse brain and in human Alzheimer brain express phosphoprotein enriched in astrocytes (PEA-15).

To identify potential biomarkers associated with Alzheimer's disease (AD)-like neuropathologies in the murine brain, we conducted proteomic analyses of neocortices from TgCRND8 mice. Here we found that phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is expressed at higher levels in the neocortical proteomes from 6-month old TgCRND8 mice, as compared to non-transgenic mice. Immunostaining for PEA-15 revealed reactive astrocytes associated with the neocortical amyloid plaques in TgCRND8 mice and in post-mortem human AD brains. This is the first report of increased phosphoprotein enriched in astrocytes (PEA-15) expression in reactive astrocytes of an AD mouse model and human AD brains.

Olfactory ensheathing cells of hamsters, rabbits, monkeys, and mice express α-smooth muscle actin.

Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system, residing in the olfactory mucosa and at the surface of the olfactory bulb. We investigated the neurochemical features of OECs in a variety of mammalian species (including adult hamsters, rabbits, monkeys, and mice, as well as fetal pigs) using three biomarkers: α-smooth muscle actin (αSMA), S100ß, and glial fibrillary acidic protein (GFAP). Mucosal and bulbar OECs from all five mammalian species express S100ß. Both mucosal and bulbar OECs of monkeys express αSMA, yet only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Mucosal OECs, but not bulbar OECs, also express GFAP in hamsters and monkeys; mice, by comparison, have only a sparse population of OECs expressing GFAP. Though αSMA immunostaining is not detected in OECs of adult mice, GFAP-expressing mucosal OECs isolated from adult mice do coexpress αSMA in vitro. Moreover, mucosal OECs from adult mutant mice lacking αSMA expression display perturbed cellular morphology (i.e., fewer cytoplasmic processes extending among the hundreds of olfactory axons in the olfactory nerve fascicles and nuclei having degenerative features). In sum, these findings highlight the efficacy of αSMA and S100ß as biomarkers of OECs from a variety of mammalian species. These observations provide definitive evidence that mammalian OECs express the structural protein αSMA (at various levels of detection), which appears to play a pivotal role in their ensheathment of olfactory axons.

Overexpression of nerve growth factor by murine smooth muscle cells: role of the p75 neurotrophin receptor on sympathetic and sensory sprouting.

Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α-actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF-sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene-related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF-rich peripheral tissues.

Changes in hepatic protein expression in spontaneously hypertensive rats suggest early stages of non-alcoholic fatty liver disease.

Hypertension is a systemic disorder affecting numerous physiological processes throughout the body. As non-alcoholic fatty liver disorder (NAFLD) is a common comorbidity of hypertension in humans, we hypothesized that molecular hepatic physiology would be altered in a model of genetic hypertension. Despite the broad use of the spontaneously hypertensive rat (SHR) model, little is known regarding how hypertension influences hepatic function under basal conditions. In order to determine whether hypertension induces changes in the hepatic protein expression suggestive of early stages of NAFLD, we compared the whole tissue proteome of livers from SHR and Wistar Kyoto (WKY) 16 week old rats using 2DGE and MALDI-TOF MS. Fifteen proteins were identified that display different levels of expression between the SHR and WKY livers: 50% of proteins have mitochondrial or anti-oxidant functions while 20% are involved in lipid metabolism. Quininoid dihydropterin reductase, sulfite oxidase, and glutathione-S-transferase mu 1 were all identified as either undergoing a difference in post-translation modification or a difference in protein abundance in SHR compared to WKY livers. As oxidative stress is a well described component of both NAFLD and hypertension in SHR, the identification of novel changes in protein expression provides possible mechanisms connecting these two pathologies in humans.

Nerve growth factor promoter activity revealed in mice expressing enhanced green fluorescent protein.

Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury.

A systematic review of cellular transplantation therapies for spinal cord injury.

Cell transplantation therapies have become a major focus in pre-clinical research as a promising strategy for the treatment of spinal cord injury (SCI). In this article, we systematically review the available pre-clinical literature on the most commonly used cell types in order to assess the body of evidence that may support their translation to human SCI patients. These cell types include Schwann cells, olfactory ensheathing glial cells, embryonic and adult neural stem/progenitor cells, fate-restricted neural/glial precursor cells, and bone-marrow stromal cells. Studies were included for review only if they described the transplantation of the cell substrate into an in-vivo model of traumatic SCI, induced either bluntly or sharply. Using these inclusion criteria, 162 studies were identified and reviewed in detail, emphasizing their behavioral effects (although not limiting the scope of the discussion to behavioral effects alone). Significant differences between cells of the same "type" exist based on the species and age of donor, as well as culture conditions and mode of delivery. Many of these studies used cell transplantations in combination with other strategies. The systematic review makes it very apparent that cells derived from rodent sources have been the most extensively studied, while only 19 studies reported the transplantation of human cells, nine of which utilized bone-marrow stromal cells. Similarly, the vast majority of studies have been conducted in rodent models of injury, and few studies have investigated cell transplantation in larger mammals or primates. With respect to the timing of intervention, nearly all of the studies reviewed were conducted with transplantations occurring subacutely and acutely, while chronic treatments were rare and often failed to yield functional benefits.

Microglial/macrophage cells in mammalian olfactory nerve fascicles.

This is the first description of a population of Iba1- and annexin A3-immunopositive cells residing in the peripheral olfactory nerves of adult rats and adult cats. Based on their ramified appearance, positive immunostaining for the monocytic markers Iba1 and annexin A3, and reactivity to bulbectomy (in adult rats), these cells found within the olfactory nerve fascicles of both mammalian species meet several important criteria for their designation as microglia/macrophages. These Iba1-/annexin A3-immunopositive cells may be uniquely positioned to protect against the potential spread of dangerous environmental xenobiotics (such as viruses and toxins) into the brain, where such pathogens may contribute to the development of neurological diseases, such Alzheimer's and Parkinson's diseases.

A comparative examination of biomarkers for olfactory ensheathing cells in cats and guinea pigs.

We investigated the neurochemical characteristics of olfactory ensheathing cells (OECs) in adult cats and in adult guinea pigs. Three conventional biomarkers for OECs, p75 neurotrophin receptor (p75NTR), S100, and glial fibrillary acidic protein (GFAP), as well as two recently identified biomarkers, smooth muscle alpha-actin (SMA) and calponin, were used. We found that 1) antibodies against SMA and S100 yielded positive immunostaining of mucosal and bulbar OECs in cats and guinea pigs; 2) antibodies against GFAP gave positive immunostaining of mucosal and bulbar OECs in cats; and 3) antibodies against calponin yielded positive immunostaining of bulbar OECs in adult cats. Unexpectedly, antibodies against p75NTR failed to positively stain mucosal and bulbar OECs in cats and guinea pigs, and antibodies against GFAP and calponin failed to positively stain mucosal and bulbar OECs in guinea pigs. These findings show the importance for empirical testing of all biomarkers for OECs among different mammalian species when attempting to identify these cells in vivo, in vitro, and following intraspinal implantation.

Transgenic mice expressing nerve growth factor in smooth muscle cells.

Ectopic expression of nerve growth factor (NGF) in transgenic mice leads to site-specific sympathetic sprouting. Smooth muscle cells in the intestines, urinary bladder, and arteries have been shown to express NGF. To address whether enhanced NGF production among these different organ systems stimulates comparable patterns of sympathetic collateral growth, we generated transgenic mice that express NGF under the control of the smooth muscle alpha-actin promoter. In response to elevated levels of NGF protein in the colon, bladder, and arteries/arterioles, sympathetic axons displayed robust sprouting only in the colon and bladder. These data reveal that, unlike most other peripheral tissues, sympathetic efferents in adult mammalian arteries/arterioles do not undergo collateral growth in response to increased levels of smooth muscle-derived NGF.

Technical strategies to isolate olfactory ensheathing cells for intraspinal implantation.

Over the past few years, the idea of using intraspinal implantations of olfactory ensheathing cells (OECs) as a therapeutic strategy to enhance recovery after spinal cord injury has quickly moved from experimentation with laboratory mammals to surgical approaches for paralyzed humans. Despite this progression, several important issues have yet to be thoroughly addressed: for instance, which of the many methods currently being used best yields enriched populations of OECs, and how such purity can be empirically tested and validated among different mammalian species, including humans. Here we offer an authoritative review of those methods used to isolate OECs from the olfactory mucosa and/or olfactory bulbs of rats, mice, dogs, pigs, non-human primates, and humans. As well, we assess which biomarkers are currently being utilized to determine the relative proportions of OECs and contaminating cells in these glial cultures. Although there have been numerous review articles regarding OECs in vitro, our review is unique in that it offers a critical assessment of the methods currently being used to generate cultures of mammalian OECs. More specifically, we examine the issue of culture contamination by phenotypically similar Schwann cells. This review is timely because recent clinical usage of OECs has come under intense criticism for a number of reasons, including the reliable identification of cultured human OECs. We believe that once these methodological issues of isolation and characterization of OECs have been resolved, this glial population will offer paralyzed individuals a truly viable cellular strategy for intraspinal therapy.

Proteomic assessment of sympathetic ganglia from adult mice that possess null mutations of ExonIII or ExonIV in the p75 neurotrophin receptor gene.

Neurotrophins, such as nerve growth factor (NGF), are capable of binding to the transmembrane p75 neurotrophin receptor (p75NTR), which regulates a variety of cellular responses including apoptosis and axonal elongation. While the development of mutant mouse strains that lack functional p75NTR expression has provided further insight into the importance of this neurotrophin receptor, there remains a paucity of information concerning how the loss of p75NTR expression may alter neural phenotypes. To address this issue, we assessed the proteome of the cervical sympathetic ganglia from two mutant lines of mice, which were compared to the ganglionic proteome of age-matched wild type mice. The ganglionic proteome of mice possessing two mutant alleles of either exonIII or exonIV for the p75NTR gene displayed detectable alterations in levels of Lamin A, tyrosine hydroxylase, and Annexin V, as compared to ganglionic proteome of wild type mice. Decreased expression of the basic isoform of tyrosine hydroxylase may be linked to perturbed NGF signaling in the absence of p75NTR in mutant mice. Stereological measurement showed significant increases in the number of sympathetic neurons in both lines of p75NTR-deficient mice, relative to wild type mice. This enhanced survival of sympathetic neurons coincides with shifts toward the more basic isoforms of Annexin V in mutant mice. This study, in addition to providing the first comparative proteomic assessment of sympathetic ganglia, sheds new light onto the phenotypic changes that occur as a consequence of a loss of p75NTR expression in adult mice.

Olfactory ensheathing cells express smooth muscle alpha-actin in vitro and in vivo.

One strategy for spinal cord repair after injury that has moved quickly from the research laboratory to the clinic is the implantation of olfactory ensheathing cells (OECs). These unique glial cells of the olfactory system have been associated with axonal remyelination and regeneration after grafting into spinalized animals. Despite these promising observations, there remains a lack of direct empirical evidence of the exact fate of OECs after intraspinal implantation, in large part because of a surprising paucity of defined biomarkers that unequivocally distinguish these cells from phenotypically similar Schwann cells. Here we provide direct neurochemical proof that OECs, both in vitro and in vivo, express smooth muscle alpha-actin. That OECs synthesize this contractile protein (and a variety of actin-binding proteins including caldesmon) provides compelling evidence that these cells are, in fact, quite different from Schwann cells. The identification of several smooth muscle-related proteins in OECs points to a new appreciation of the structural and functional features of this population of olfactory glia. These biomarkers can now be used to elucidate the fate of OECs after intraspinal implantation, in particular assessing whether smooth muscle alpha-actin-expressing OECs are capable of facilitating axon remyelination and regeneration.

Neurotrophic activity of proNGF in vivo.

Ectopic expression of nerve growth factor (NGF) in transgenic mice results in the directional growth of sympathetic and/or sensory fibers. For instance, mice that over-express NGF under the control of the glial fibrillary acidic protein (GFAP) promoter exhibit robust axonal sprouting into the cerebellum, with no apparent loss of neurons in peripheral ganglia. Given the disagreement in the literature over whether pro-NGF exerts neurotrophic or apoptotic effects, we assessed the relative levels of proNGF and mature NGF in the cerebella of these transgenic mice. Blinded western blot analyses revealed that proNGF was the major species in both transgenic and wild type mice, with very low levels of mature NGF expression. While transgenic mice displayed significantly higher levels of cerebellar proNGF protein as compared to wild type mice, both strains possessed comparable levels of mature NGF. These data reveal that the ectopic expression of NGF in the cerebellum results in an increase in proNGF rather than mature NGF levels. Together with the robust axonal growth and lack of neuronal death in the ganglia in these animals, our results are clearly consistent with proNGF exhibiting neurotrophic activity in vivo.

Cultures of rat olfactory ensheathing cells are contaminated with Schwann cells.

Implantation of cultured olfactory ensheathing cells into the damaged spinal cord of adult rats has been reported to remyelinate central axons. This observation is curious because olfactory ensheathing cells do not myelinate axons in their native environment. We have recently determined that calponin is the first definitive phenotypic marker for olfactory ensheathing cells. Primary cultures of adult rat olfactory mucosa and olfactory bulb were immunostained for p75 neurotrophin receptor and calponin. Our results reveal that two populations of p75 neurotrophin receptor-positive cells exist in primary cultures of the olfactory mucosa and bulb: calponin-positive olfactory ensheathing cells and calponin-negative Schwann cells. As olfactory tissues likely yield a mixed glial population, the idea that olfactory ensheathing cells are capable of de novo myelin synthesis after intraspinal implantation should be re-evaluated.

Null mutations for exon III and exon IV of the p75 neurotrophin receptor gene enhance sympathetic sprouting in response to elevated levels of nerve growth factor in transgenic mice.

Under normal conditions, expression of the p75 neurotrophin receptor (p75NTR) by sympathetic neurons can increase the affinity of the signaling receptor, trkA, to target-derived nerve growth factor (NGF) at distal axons. We have previously reported that sprouting of sympathetic axons into NGF-rich target tissues is enhanced when p75NTR expression is perturbed, leading to the postulate that p75NTR may restrain sympathetic sprouting in response to elevated NGF levels. These observations were made using mice having a null mutation of the third p75NTR exon, a line that may express a hypomorphic form of this receptor. Since mice carrying a null mutation of the fourth p75NTR exon may not express a similar splice variant, we sought to determine whether these animals possess the same phenotype of enhanced sympathetic sprouting in response to elevated levels of NGF. Both lines of transgenic mice lacking p75NTR displayed similar degrees of sympathetic axonal sprouting into the cerebellum and trigeminal ganglia, two target tissues having elevated levels of NGF protein. Furthermore, the densities of sympathetic axons in both targets were significantly greater than those observed in age-matched NGF transgenic siblings expressing full-length p75NTR. Our new findings provide a comparative analysis of the phenotype in two independent mutations of the same neurotrophin receptor, revealing that p75NTR plays an important role in restricting sympathetic sprouting in response to higher NGF levels.