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Extracellular vesicles (EVs) are released from many cell types, including normal and pathological cells, and range from 30 to 1000 nm in size. Once thought to be a mechanism for discarding unwanted cellular material, EVs are now thought to play a role in intercellular communication. Evidence is accruing that EVs are capable of carrying mRNAs, miRNAs, noncoding RNAs, and proteins, including those associated with neurodegenerative diseases and cancer, which may be exchanged between cells. For this reason, neurodegenerative diseases and cancers may share a common mechanism of disease spread via EVs. Understanding the role EVs play in disease initiation and progression will aid in the discovery of new clinically relevant biomarkers and the development of better targeted molecular and biological therapies.

Identifying novel, effective therapeutics for Alzheimer's disease (AD) is one of the major unmet medical needs for the coming decade. Because the current paradigm for developing and testing disease modifying AD therapies is protracted and likely to be even longer with the shift towards earlier intervention in pre-clinical AD, it is an open question whether we can develop, test, and widely deploy a novel therapy in time to help the current at-risk generation if we continue to follow the standard paradigms of discovery and drug development. There is an imperative need to find safe and effective preventative measures that can be rapidly deployed to stem the coming wave of AD that will potentially engulf the next generation. We can broadly define regenerative medicine as approaches that use stem-cell-based therapies or approaches that seek to modulate inherent neurogenesis. Neurogenesis, though most active during pre-natal development has been shown to continue in several small parts of the brain, which includes the hippocampus and the subventricular zone, suggesting its potential to reverse cognitive deficits. If AD pathology impacts neurogenesis then it follows that conditions that stimulate endogenous neurogenesis (e.g., environmental stimuli, physical activity, trophic factors, cytokines, and drugs) may help to promote the regenerative and recovery process. Herein, we review the complex logistics of potentially implementing neurogenesis-based therapeutic strategies for the treatment of AD.

The presence of stem and progenitor cells in the adult human brain suggests a putative and persistent role in reparative behaviors following neurological injury and neurological disease. Too few stem/progenitor cells (as in the case of Parkinson's disease) or too many of these cells (as in the case of Huntington's disease and glioma) could contribute to and even signal brain pathology. We address here critical issues faced by the field of stem cell biology and regenerative medicine, arguing from well-documented as well as speculative perspectives for a potential role for stem cells in the pathology of many human neurological diseases. Although stem cell responses may result in regenerative failure, in many cases they may help in the establishment or re-establishment of a functional neural circuitry (eg, after stroke). Therefore, we would argue that stem cells have a crucial-either positive or negative-role in the pathology of many neurological diseases.

Cerebellar Purkinje neurons (PNs) possess a well characterized propensity to fuse with bone marrow-derived cells (BMDCs), producing heterokaryons with Purkinje cell identities. This offers the potential to rescue/repair at risk or degenerating PNs in the inherited ataxias, including Spinocerebellar Ataxia 1 (SCA1), by introducing therapeutic factors through BMDCs to potentially halt or reverse disease progression. In this study, we combined gene therapy and a stem cell-based treatment to attempt repair of at-risk PNs through cell-cell fusion in a Sca1(154Q/2Q) knock-in mouse model. BMDCs enriched for the hematopoietic stem cell (HSC) population were genetically modified using adeno-associated viral vector 7 (AAV7) to carry SCA1 modifier genes and transplanted into irradiated Sca1(154Q/2Q) mice. Binucleated Purkinje heterokaryons with sex-mismatched donor Y chromosomes were detected and successfully expressed the modifier genes in vivo. Potential effects of the new genome within Purkinje heterokaryons were evaluated using nuclear inclusions (NIs) as a biological marker to reflect possible modifications of the SCA1 disease process. An overall decrease in number of NIs and an increase in the number of surviving PNs were observed in treated Sca1(154Q/2Q). Furthermore, Bergmann glia were found to have fusogenic potential with the donor population and reveal another potential route of therapeutic entry into at-risk cells of the SCA1 cerebellum. This study presents a first step towards a proof-of-principle that combines somatic cellular fusion events with a neuroprotective gene therapy approach for providing potential neuronal protection/repair in a variety of neurodegenerative disorders.

Slice culture model systems provide a unique opportunity to monitor and lesion brain circuits in a dish. Using a novel approach, we have generated parasagittal slices from mouse brains that preserve, throughout the culture process, the nigrostriatal circuit. These slices can be cultured for approximately 4 weeks with maintenance of normal neuronal cytoarchitecture. Application of the dopamine specific toxin 6-hydroxy dopamine (6-OHDA) induces a significant decline in tyrosine hydroxylase positive cell bodies and fibers. Using a transgenic mouse with green fluorescent protein (GFP) under the control of the tyrosine hydroxylase promoter, we have been able to visualize in real time the loss of GFP expression in the striatum of slices as a result of 6-OHDA exposure. Using these cultures we have demonstrated the feasibility of modeling cellular replacement strategies. GFP-positive embryonic stem cell-derived neuronal precursors can be tracked in real time throughout the experiment and are amenable to patch clamp recording within the slice environment. In addition, cell differentiation can be observed within these slices and the effects of morphogenetic proteins, like the extracellular matrix molecule laminin, drugs or small molecules can be observed. This unique culture system presents a new approach for modeling Parkinson's disease in vitro, and provides a potentially useful new method for screening cell and molecular therapies for neurodegenerative diseases.

Hypoxic-ischemic encephalopathy (HIE) in neonates results in long-term disabilities. Stem cell therapy may offer an attractive treatment for HIE. Multipotent astrocytic stem cells (MASCs) from mice transplanted into a rat model of hypoxia-ischemia (HI) survived the transplantation and showed signs of migration towards the injured cortex. Some MASCs around the injured cortex differentiated into neuronal and astrocytic phenotypes. MASCs transplanted into non-ischemic pups survived but retained their astrocytic phenotype. These data suggest that transplanted MASCs can survive and differentiate into neurons and astrocytes in the post-injury milieu of the neonatal brain injured by HI.

Brain slices provide a useful nervous tissue model to investigate the relationships between magnetic resonance imaging (MRI) contrast mechanisms and tissue microstructure; yet, these acutely isolated tissues remain viable for only 10–12 h. To study slower biological processes, this work describes the first MRI microscopy characterization of organotypic rat hippocampal slice cultures that can be maintained for several weeks. Diffusion-weighted images of slice cultures acquired with a 14.1-T magnet demonstrated the laminar anatomy of the hippocampus with relatively high signal-to-noise ratios. Diffusion data analyzed using a two-compartment model with exchange indicated that cultured slices had a comparable microstructure to acute brain slices and to in vivo brain. Immunohistochemistry indicated that slice cultures tolerated the conditions required for MRI study well. MRI of cultured tissue slices is highly amenable to correlative microscopy techniques and offers great promise for future MRI investigations of pathological tissue reorganization, molecular imaging and stem cell therapies.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.

Summary Background End-organ repair by adult haemopoietic stem cells is under great scrutiny with investigators challenging the notion of these cells' plasticity. Some investigations of animals and short-term human bone marrow transplants suggest that bone marrow can repair brain. We looked for evidence of clinically relevant marrow-derived restorative neurogenesis: long-term, multilineage, neural engraftment that is not the result of cell-fusion events. Methods We examined autopsy brain specimens from three sex-mismatched female bone-marrow-transplantation patients, a female control, and a male control. We did immunohistochemistry, fluorescence in-situ hybridisation, and tissue analysis to look for multilineage, donor-derived neurogenesis. Findings Hippocampal cells containing a Y chromosome were present up to 6 years post-transplant in all three patients. Transgender neurons accounted for 1% of all neurons; there was no evidence of fusion events since only one X chromosome was present. Moreover, transgender astrocytes and microglia made up 1–2% of all glial cells. Interpretation Postnatal human neuropoiesis happens, and human haemopoietic cells can transdifferentiate into neurons, astrocytes, and microglia in a long-term setting without fusing. Transplantable human haemopoietic cells could serve as a therapeutic source for long-term regenerative neuropoiesis.

There is a paucity of information on the roles of extracellular matrix (ECM) and substrate molecules in general with regard to the growth and differentiation of neural stem and progenitor cells. There are well-established findings of a dense, presumably astrocyte-derived ECM in the persistently neurogenic subependymal zone and its migratory extension the rostral migratory stream. Cells cultured from this region, as well as from early postnatal cerebellum, generate multipotent neurospheres, but at present there is little information as to the ECM regulation of these neural stem cell populations. The present study examined the behavior of cerebellar-derived neurospheres on the matrix components laminin, fibronectin, and chondroitin sulfate proteoglycan. The results showed that laminin and fibronectin significantly increase cell migration velocity as compared to CSPG. Fibronectin effected a maximal velocity after 48 h, whereas maximal velocity on laminin and CSPG was not reached until 72 h. Both laminin and fibronectin were very permissive substrates for cellular outgrowth. Chondroitin sulfate proteoglcyan showed a significant inhibition of migratory outgrowth and velocity. These ECM molecules did not appear to affect the fate choice of neurons and glia, thus their role in neuropoietic structures may be to facilitate or deter cell movement and process outgrowth.

Stem cells in adult tissues have attracted a great deal of interest. These cells are self-renewing and can give rise to diverse progeny. An extraordinary finding was the presence of stem cells in the mature human brain. This tissue was previously believed incapable of generating new neurons, but neuropoiesis is now an established phenomenon in the adult brains of mammals, including human beings. This persistent neurogenesis has potential therapeutic applications for various neurological disorders as a source for tissue engraftment and as self-repair by a person's own indigenous population of pluripotent cells or biogenic by-products of their proliferation and differentiation.

The adult mammalian CNS harbors a population of multipotent stem/progenitor cells that can be induced to grow as proliferative neurospheresin vitro.We demonstrate here that neurosphere-generating cells can be isolated from adult mouse spinal cord and forebrain subependymal zone after postmortem intervals of up to 140 h, when kept at 4°C, and up to 30 h when kept at room temperature. Although there was an inverse relationship between postmortem interval and the number of neurospheres generated, neurospheres derived under these conditions were proliferative and could give rise to both neurons and glia.

Previous studies describing the use of cryoculture methods have focused on the efficacy of the method for studying neuron attachment and neurite outgrowth on intact sections of nerve, and rodent and even human brain. The cryoculture method has shown promise for determining the presence of cell attachment- and neurite-growth-inhibiting molecules in such specimens, and some studies have also attempted to neutralize such molecules with antibodies to myelin inhibitory proteins, nerve growth factor, or factors present in conditioned media that may counteract the repulsiveness of some of these molecules preserved in sections of, for example, myelinated nerves or adult brain white matter. The present study describes the novel use of lesioned central nervous system cryocultures as substrates for investigating the attachment of embryonic neurons and PC12 cells. In addition to demonstrating the use of this novel scar substrate to extend previous ‘scar-in-a-dish’ models (David et al. (1990) Neuron, 5: 463–469; Rudge and Silver (1990) J. Neurosci., 10: 3594–3603; Rudge et al. (1989) Exp. Neurol., 103: 1–16), the present study also describes antibody and lectin perturbations of putative inhibitory molecules that result in an enhanced attachment of cells to cryosection cultures of brain and spinal cord wounds.

Deoxyribonucleic acid of cells undergoing apoptosis is cleaved by a calcium-dependent endonuclease into oligonucleosomal-sized fragments. These fragments can be labeled using the enzyme terminal deoxynucleotidyl transferase so that the cells can be visualized immunohistochemically. Few investigators have evaluated this method in disease processes of the human central nervous system. The Tdt-mediated dUTP-biotin nick end labeling (TUNEL) technique has been investigated in preliminary studies of a variety of pathologic conditions of the human brain (e.g., gliomas, traumatic brain injury, Parkinson's disease, Parkinson's-Alzheimer's complex, multisystem atrophy, striatonigral degeneration). We focus, however, on Huntington's disease (HD) because of the availability of well-characterized pathological stages for study, and also because of the neurodegenerative diseases studied to date, only Huntington's disease revealed significant and consistent labeling with this method. This implies a possibly unique nature to the mechanism of cell death in Huntington's disease compared to the other neurodegenerative diseases studied. TUNEL+ neurons were found in Grade 1-4 HD neostriatum, while labeled astrocytes were found predominantly in the Grade 1 and 2 cases studied to date. TUNEL+ cells were also found in glioblastoma multiforme and traumatic brain injury. We conclude that while there appear to be several limitations associated with this technique, it may be useful for identifying both apoptosis and necrosis in certain neuropathological conditions.

Certain types of glial structures, located at strategic positions along axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development. The roof plate, a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons. We examined the roof plate to determine the developmental morphology of the region and to determine which molecules were correlated with the barrier function when axons were growing nearby. Light and electron microscopic observations of the roof plate revealed that this glial domain undergoes a dramatic change in shape from a “wedge” with large extracellular spaces between the cell apices at E12.5 to a thin, dense septum with reduced extracellular space at E15.5. Immunocytochemical techniques demonstrated that highly sialylated neural cell adhesion molecule (N-CAM), the carbohydrate recognized by L2 monoclonal antibody, cholinesterase, stage-specific embryonic antigen 1, and a ligand that binds tetragonolobus purpureas agglutinin are expressed by the roof plate. These molecules, however, were also found in other regions of the spinal cord which are permissive or attractive to axon growth. A molecule which is unique to the roof plate when axons grow close to, but do not cross, the dorsal midline is a glycosaminoglycan (GAG), keratan sulfate. Keratan sulfate is also present in the tectal midline and in other noninnervated regions such as the outer epidermis and developing cartilage. Our data suggest that keratan sulfate, alone or in combination with other molecules expressed by the roof plate, may be responsible, in part, for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.

The present study demonstrates that the boundary patterns of cell surface-associated molecules detected with lectins in the barrel cortex of neonatal rodents are altered, as are the boundary patterns of cortical glia, following perturbation of large vibrissae in the contralateral mystacial face pad. The alterations in the transiently expressed molecular patterns of lectin-receptors provide data that are consistent with the idea that the periphery plays a prominent role in the establishment of functional cytoarchitecture in the developing cortex. The data are also consistent, however, with the notion that factors intrinsic to the cerebrum, such as the immature cortical glial cells, are of considerable importance in this respect and a direct or indirect interaction of thalamocortical afferents with glial cells in the somatosensory cortex of the neonate are indicated. It is suggested therefore that a critical period in early barrel development, a time in which the cortical neuronal architecture is malleable in response to altered afferent input, is directly related to the presence of these cellular and molecular boundaries. The transient barrel boundaries, it is argued, are the morphological and molecular substrates that form the physical basis of the critical period.

The localization of glycosylated molecules and glia has been studied during early postnatal development in the mouse central nervous system (CNS) using autoradiographic detection of radiolabeled fucose incorporation, and in sections processed either for histochemistry or immunocytochemistry following binding of labeled lectins or an antibody to glial fibrillary acidic protein. Radiolabeled sugar incorporation, lectin binding of glycoconjugates, and glial labeling all reveal borders between nuclei within the diencephalon, midbrain, and brainstem through the first postnatal week. Glycoconjugate and glial boundaries exist throughout the CNS during pattern formation events, and they also are seen in relation to fine aspects of developing functional organization within individual structures (e.g. segmentation associated with the representation of mystacial vibrissae within the brainstem trigeminal complex). The observation that each of the probes employed in this study fails to label boundary organization during later postnatal times suggests that the distribution and chemistry of the glial/glycoconjugate network are dynamic, and they change in accordance with distinct maturational states of the nervous system.

Retrogradely labeled neurons were found in the spinal trigemial (Spin V.) and principal sensory nucleus (Prin V.) and other regions following injections of horseradish peroxidase (HRP) into normal mouse cerebellar hemisphere and vermis. HRP injections into particular regions of the malformed cerebellum in the reeler mouse also resulted in retrograde labeling within regions of the trigeminal complex that are topographically similar to those seen in the normal. In association with a recently described cerebellar tactile representation of the head [17], the findings suggest that the neurons of origin of this somatosensory projection reside in portions of the trigeminal complex and possibly in other brainstem nuclei, in both normal mice and reeler mutants. These data also contribute to an understanding of afferent organization and the maintenance of appropriate patterns of connectivity within the malformed reeler cerebellum.

During early postnatal development in reeler mutant mice, lectin binding delineates prospective abnormal barrels as they will appear in the adult mutant somatosensory cortex. Glial fibers also may be more condensed within fascicles in developing reeler barrels. These fibers also appear to be misaligned, coursing predominantly in the tangential plane within the abnormal reeler barrel sides as opposed to having a radial orientation as seen in normal mouse barrels. The thalamic barreloid complex, however, reveals a disposition of glycoconjugates that is completely normal in reeler. Thus, there are anomalies in glia and associated glycoconjugates during mainly cortical development in the reeler mutant mouse that might be related to the primary action of the abnormal gene.

Utilizing a recently introduced double retrograde axonal tracing technique, the existence of collateralized axonal projections of locus coeruleus neurons to both forebrain and cerebellar cortical structures has been examined in adult mice. Following paired injection strategies using horseradish peroxidase and radiolabeled wheat germ agglutinin within portions of the forebrain and cerebellar cortices, substantial numbers of double labeled ‘branched’ neurons have been found that reside within all regions og the nucleus.