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Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10μM), or large inclusions (≥10μM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Parkinson's disease is characterized and diagnosed by bradykinetic motor symptoms caused by the loss of dopamine neurons in the substantia nigra. The pathological and non-motor behavioral changes that occur prior to degeneration are less well characterized, although changes in gait, olfaction and cognition have been recognized in familial Parkinson's disease subjects. Gene mutations associated familial Parkinson's disease give rise to mitochondrial changes, altered energy homeostasis and intracellular trafficking deficits, and these can be modeled in transgenic mice. Here we discuss the recent finding of prodromal behavioral disturbances in a PINK1 deficient mouse that manifest prior to dopaminergic cell death and correlate to 5-HT fiber losses and mitochondrial morphological changes. We discuss the representation of the PINK1 deficient mouse and other genetic models to accurately recapitulate early Parkinson's disease. Prodromal symptoms and underlying pathology modeled in mice and cell lines from human subjects may have wide implications for earlier diagnosis. Current and emerging therapies need to be tailored to target both early cognitive and late stage motor symptoms., (Published by Elsevier Inc.)

We evaluated Ts65Dn Down's syndrome mice and their littermates (LM) at 1-2, 4, and 12 months of age to determine amyloid precursor protein (APP)-related cellular and biochemical changes associated with cognitive deficits. Ts65Dn mice showed cognitive deficits in the Morris water maze compared to LM mice at 4 and 12 months of age. Ts65Dn, but not LM mice, developed a septohippocampal cholinergic neuronal degeneration of choline acetyltransferase (ChAT)-positive neurons at 12 months of age. These cellular changes were compensated by increases in ChAT enzyme activity of remaining cholinergic terminals in the hippocampus. By 12 months of age, Ts65Dn mice had elevations of APP protein levels in the hippocampus compared to their LM. At this age, both Ts65Dn mice and their LM abnormally responded to cholinergic muscarinic M1 agonist treatment in terms of hippocampal APP, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) levels compared to young adult C57BL/6 mice. In summary, the Ts65Dn mice show developmental and progressive age-related behavioral deficits, hippocampal APP, and cholinergic pathology. The relatively better cognitive spatial performance in LM compared to Ts65Dn mice suggests that high APP levels combined with progressive degeneration of the cholinergic system are critical to the pathology and cognitive deficits seen in Ts65Dn mice.

In the present study, we have characterized an atraumatic grafting technique which permits multiple, segmental, and lamina-specific injections into cervical or lumbar spinal cord. Cell injections were performed in spinally mounted rats of different ages and spinal cord size, using a micromanipulator and glass microcapillary connected to a digital microinjector. For grafting, we used human neuroteratoma (hNT) cells, BrdU-labeled rat spinal precursors or primary embryonic spinal cord neurons isolated from E14 spinal cord of the eGFP+ rat. Systematic quantification of grafted cells was performed using stereological principles of systematic random sampling and semi-automated optical Disector software. Volume reconstruction was performed using serial sections from grafted areas and custom-developed software (Ellipse) which permits "two reference points" semi-automated alignment of images, as well as volume reconstruction and calculation. By coupling these techniques, it is possible to achieve a relatively precise and atraumatic cell delivery into multiple spinal cord segments and specific spinal laminae. Consistency of the multiple grafts position in the targeted laminar areas was verified by a systematic volume reconstruction. Good survival of implanted cells for the three different cell lines used indicate that this grafting technique coupled with a systematic analysis of the individual grafting sites can represent a valuable implantation-analytical system.

Parkinson's disease (PD) is characterized pathologically by preferential degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). Nigral cell death is accompanied by the accumulation of a wide range of poorly degraded proteins and the formation of proteinaceous inclusions (Lewy bodies) in dopaminergic neurons. Mutations in the genes encoding alpha-synuclein and two enzymes of the ubiquitin-proteasome system, parkin and ubiquitin C-terminal hydrolase L1, are associated with neurodegeneration in some familial forms of PD. We now show that, in comparison to age-matched controls, alpha-subunits (but not beta-subunits) of 26/20S proteasomes are lost within dopaminergic neurons and 20S proteasomal enzymatic activities are impaired in the SNc in sporadic PD. In addition, while the levels of the PA700 proteasome activator are reduced in the SNc in PD, PA700 expression is increased in other brain regions such as the frontal cortex and striatum. We also found that levels of the PA28 proteasome activator are very low to almost undetectable in the SNc compared to other brain areas in both normal and PD subjects. These findings suggest that failure of the ubiquitin-proteasome system to adequately clear unwanted proteins may underlie vulnerability and degeneration of the SNc in both sporadic and familial PD.

The physiological interrelationships between cognitive impairments, neurotransmitter loss, amyloid processing and energy metabolism changes in AD, cholinergic dementia and Down's syndrome are largely unknown to date. This report contains novel studies into the association between cognitive function and cerebral metabolism after long-term selective CNS cholinergic neuronal and synaptic loss in a rodent model. We measured local cerebral rates of glucose utilization ((14)C-2-deoxyglucose) throughout the brains of awake rats 4.5 months after bilateral intraventricular injections of a cholinotoxic antibody directed against the low-affinity NGF receptor (p75 NGF) associated with cholinergic neurons (192 IgG-saporin). Permanent cholinergic synapse loss was demonstrated by [(3)H]-vesamicol in vitro autoradiography defining presynaptic vesicular acetylcholine (ACh) transport sites. While other metabolic studies have defined acute and transient glucose use changes after relatively nonspecific lesions of anatomical regions containing cholinergic neurons, our results show sustained reductions in glucose utilization in brain regions impacted by cholinergic synapse loss, including frontal cortical and hippocampal regions, relative to glucose use levels in control rats. In the same animals, impaired cognitive spatial performance in a Morris water maze was correlated with reduced glucose use rates in the cortex and hippocampus at this time point, which is consistent with increased postmortem cortical and hippocampal amyloid precursor protein (APP) levels (45, 46). These results are consistent with the view of cholinergic influence over metabolism, APP processing, and cognition in the cortex and hippocampus., (Copyright 2001 Academic Press.)

The elucidation of the genetic defect in patients with Huntington's disease (HD) has allowed for the detection of individuals at risk for HD prior to the onset of symptoms. Thus "neuroprotection strategies" aimed at preventing the neuropathological and behavioral sequelae of this disease might be powerful therapeutically since they could be introduced to healthy patients before the initiation of a massive degenerative cascade principally localized to the striatum. A variety of trophic factors potently protect vulnerable striatal neurons in animal models of HD. A number of experimental variables are critical in determining the success of trophic factors in animal models. In this regard, the method of trophic factor delivery may be crucial, as delivery via genetically modified cells often produces greater and more widespread effects on striatal neurons than infusions of that same factor. The mechanisms by which cellularly delivered trophic factors forestall degeneration and prevent behavioral deficits are complex and often appear to be unrelated to the trophic factor binding to its cognate receptor. In this regard, cells genetically modified to secrete nerve growth factor (NGF) or ciliary neurotrophic factor (CNTF) protect degenerating striatal neurons which do not express either NGF or CNTF receptors. This review will discuss some of the non-receptor-based events that might underlie these effects and present the hypothesis that cellular delivery of certain trophic factors using genetically modified cells may be ready for clinical testing in HD patients., (Copyright 1999 Academic Press.)

Fetal ventral mesencephalic (VM) transplants have been studied in the context of dopaminergic (DA) replacement therapy for Parkinson's disease (PD). DA neurons from VM transplants will grow axons and form functional synapses in the adult host central nervous system (CNS). Recently, studies have demonstrated that most of the transplanted DA neurons die in grafts within the first week after implantation. An important feature of neural development, also in transplanted developing fetal neural tissue, is cell death. However, while about 50% of cells born in the CNS will die naturally, up to 99% of fetal cells die after neural transplantation. It has been shown that VM grafts contain many apoptotic cells even at 14 days after transplantation. The interleukin-1beta converting enzyme (ICE) cysteine protease and 11 other ICE-like-related proteases have been identified, now named caspases. Activation of caspases is one of the final steps before a neuron is committed to die by apoptosis. Here we review this cell death process in detail: Since the growth of fetal neural grafts placed in the adult brain in many ways mimics normal development, it is likely that the caspases also play a functional role in transplants. Pharmacological inhibitors of caspases and genetically modified mice are now available for the study of neuronal death in fetal neuronal transplants. Understanding cell death mechanisms involved in acute cellular injury, necrosis, and programmed cell death (PCD) is useful in improving future neuronal transplantation methodology, as well as in neuroprotection, for patients with neurodegenerative diseases., (Copyright 1999 Academic Press.)

In order to assess the potential of embryonic stem cells to undergo neuronal differentiation in vivo, totipotent stem cells from mouse blastocysts (D3 and E14TG2a; previously expanded in the presence of leukemia inhibitory factor) were transplanted, with or without retinoic acid pretreatment, into adult mouse brain, adult lesioned rat brain, and into the mouse kidney capsule. Intracerebral grafts survived in 61% of cyclosporine immunosuppressed rats and 100% of mouse hosts, exhibited variable size and morphology, and both intracerebral and kidney capsule grafts developed large numbers of cells exhibiting neuronal morphology and immunoreactivity for neurofilament, neuron-specific enolase, tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and cells immunoreactive for glial fibrillary acidic protein. Though graft size and histology were variable, typical grafts of 5-10 mm3 contained 10-20,000 TH+ neurons, whereas dopamine-beta-hydroxylase+ cells were rare. Most grafts also included nonneuronal regions. In intracerebral grafts, large numbers of astrocytes immunoreactive for glial fibrillary acidic protein were present. Both TH+ and 5-HT+ axons from intracerebral grafts grew into regions of the dopamine-lesioned host striatum. TH+ axons grew preferentially into striatal gray matter, while 5-HT+ axons showed no white/gray matter preference. These findings demonstrate that transplantation to the brain or kidney capsule can induce a significant fraction of totipotent embryonic stem cells to become putative dopaminergic or serotonergic neurons and that when transplanted to the brain these neurons are capable of innervating the adult host striatum.

Diseases resulting from defects in a single gene may be more amenable to treatment by conventional gene therapy strategies than idiopathic or polygenic disorders. We have attempted to reduce the expression in vivo of the Huntington's disease gene protein, Huntingtin, using an 18-mer fluorescein-labeled phosphorothiorated antisense oligodeoxynucleotide (ODN) targeted against the start site of the first exon of the IT15 gene. Animals were given repeated intrastriatal infusions (5 microliters of a 100 nmol/microliter solution daily over 4 days) of the antisense ODN. The treatments ended on Day 5 and the tissue was processed for immunohistochemical and Western blot analysis. The fluorescein-labeled ODN appeared to penetrate several cells and did not cause any obvious toxicity to the neurons. The average reduction in levels of Huntingtin (16.9 +/- 7.2%) did not differ significantly between striatal tissue of antisense ODN-treated animals compared to those treated with a sense ODN or vehicle. Improved methods for molecular modifications of the IT15 gene may be needed for therapeutic initiatives.

Neurotransplantation of human fetal dopamine (DA) neurons is currently being investigated as a therapeutic modality for Parkinson's disease (PD). However, the practical limitations of human fetal transplantation indicate a need for alternative methodologies. Using the 6-hydroxydopamine rat model of PD, we transplanted dopaminergic neurons derived from Embryonic Day 27 porcine fetuses into the denervated striatum of cyclosporine-A (CyA)-treated or non-CyA-treated rats. Functional recovery was assessed by amphetamine-induced rotation, and graft survival and morphology were analyzed using neuronal and glial immunostaining as well as in situ hybridization with a porcine repeat element DNA probe. A significant, sustained reduction in amphetamine-induced rotational asymmetry was present in the CyA-treated rats whereas the non-CyA-treated rats showed a transient behavioral recovery. The degree of rotational recovery was highly correlated to the number of surviving transplanted porcine dopaminergic neurons. TH+ neuronal survival and graft volume were significantly greater in the CyA-treated group as compared to the non-CyA group. By donor-specific neuronal and glial immunostaining as well as donor-specific DNA labeling, we demonstrate that porcine fetal neuroblasts are able to survive in the adult brain of immunosuppressed rats, mediate functional recovery, and extensively reinnervate the host striatum. These findings suggest that porcine DA neurons may be a suitable alternative to the use of human fetal tissue in neurotransplantation for PD.

There may be important relationships between glutamate receptor activation and neurotoxicity in neurodegenerative diseases. Previous experiments using cultured neurons have demonstrated a correlation between the metabolic status of neurons and their sensitivity to glutamate receptor-mediated cytotoxicity (Novelli et al. Brain Res. 451, 205, 1988). To examine such a relationship in vivo, we first established a dose-response curve for N-methyl-D-aspartate (NMDA)-induced neuronal death in the rat striatum. We then examined the interaction between metabolic impairment and infusion of NMDA at a dose below the threshold for neurotoxicity. Metabolic impairment was induced by intraperitoneal delivery of 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex II (succinic dehydrogenase). Twelve hours after 3-NP delivery we performed stereotactic infusion of NMDA or vehicle into the striatum. During mitochondrial impairment, a relatively nonneurotoxic dose of NMDA (15 nmol) produced a lesion that was significantly larger than that caused by this dose under normal metabolic conditions. At a dose normally below the threshold for neurotoxicity, metabolic impairment significantly increased the likelihood of neuronal death in the striatum by a factor of 5. Lesions were characterized by neuronal loss with gliosis and sparing of traversing fiber bundles. These results demonstrate that metabolic impairment reduces the threshold for glutamate receptor-mediated neurotoxicity in vivo. This potentiation may have implications for understanding the role of "neuronal stress" produced by glutamate receptor activation in neurodegenerative diseases and normal aging.