Scholarly article on topic 'Amyotrophic lateral sclerosis: Is the spinal fluid pathway involved in seeding and spread?'

Amyotrophic lateral sclerosis: Is the spinal fluid pathway involved in seeding and spread? Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Richard Smith, Kathleen Myers, John Ravits, Robert Bowser

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder manifested primarily by loss of upper and lower motor neurons. Current explanations for disease progression invoke regional spread attributed to the transfer of pathogenic factors among physically contiguous neurons. However, this explanation incompletely explains certain clinical and in vitro data. Considering this, we propose that the cerebrospinal fluid (CSF) pathway is likely to be a key vector for seeding local and distal disease. Subsequent disease progression would be expected to occur independently via either axonal or CSF transmission. If one accepts the hypothesis that the CSF pathway is involved in ALS progression, it follows that the choroid plexus (CP) might well be a driver of the disease process. In support of this, we briefly review the anatomical and physiological features of the CSF pathway and the choroid plexus responsible for secreting CSF. In addition, we draw attention to the interface of the CP and CSF with the immune system. We then summarize both clinical and cell culture research that supports a key role of the CSF in the establishment and inter-neuronal spread of ALS, and which suggest directions for translational research.

Academic research paper on topic "Amyotrophic lateral sclerosis: Is the spinal fluid pathway involved in seeding and spread?"

medical hypotheses

Accepted Manuscript

Amyotrophic Lateral Sclerosis: Is the Spinal Fluid Pathway Involved in Seeding and Spread?

Richard Smith, Kathleen Myers, John Ravits, Robert Bowser


Reference: To appear in:

S0306-9877(15)00278-9 http://dx.doi.Org/10.1016/j.mehy.2015.07.014 YMEHY 7990

Medical Hypotheses

Received Date: Accepted Date:

28 May 2015 16 July 2015

Please cite this article as: R. Smith, K. Myers, J. Ravits, R. Bowser, Amyotrophic Lateral Sclerosis: Is the Spinal Fluid Pathway Involved in Seeding and Spread?, Medical Hypotheses (2015), doi: 2015.07.014

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Title: Amyotrophic Lateral Sclerosis: Is the Spinal Fluid

Pathway Involved in Seeding and Spread?

■ jf

Contributing Authors: Richard Smith M.D. , Kathleen Myers

Ph.D.1, John Ravits M.D.2, Robert Bowser Ph.D.3

Corresponding Author: Richard Smith M.D.

Center for Neurologic Study, 7590 Fay Ave., Suite 517, La Jolla, CA 92037; phone 858-455-5463; fax 858-455-5464; 2

University of California San Diego School of Medicine, Dept. of Neurosciences,

9500 Gilman Dr. #0624, La Jolla, CA 92093

Barrow Neurological Institute, Gregory W. Fulton ALS and Neuromuscular Research Center, 350 West Thomas Rd., Phoenix, AZ 85013

RS i rts a grant from the Amyotrophic Lateral Sclerosis Association as well as

>onal fees from Isis Pharmaceuticals. RB reports grants from NIH/NINDS IIH NS061867 and NS068179) as well as funding from Iron Horse Diagnostics, Inc., and from Knopp Biosciences, LLC.


Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder manifested primarily by loss of upper and lower motor neurons. Current explanations for disease progression invoke regional spread attributed to the transfer of pathogenic factors amongst physically contiguous neurons. However, this explanation incompletely explains certain clinical and in vitro data. Considering this, we propose that the cerebrospinal fluid (CSF) pathway is likely to be a key vector for seeding local and distal disease. Subsequent disease progression would be expected to occur independently via either axonal or CSF transmission.

If one accepts the hypothesis that the CSF pathway is involved in ALS progression, it follows that the choroid plexus (CP) might well be a driver of the disease process. In support of this, we briefly review the anatomical and physiological features of the CSF pathway and the choroid plexus responsible for secreting CSF. In addition, we draw attention to the interface of the CP and CSF with the immune system. We then summarize both clinical and cell culture research that supports a key role of the CSF in the establishment and inter-neuronal spread of ALS, and which suggest directions for translational research.


Since the first gene for familial amyotrophic lateral sclerosis (ALS) was identified in 1993, there has been a scientific renaissance in our understanding of this disorder. To date, approximately 40 genes have been identified that contribute to the disease, including single dominantly-inherited genes producing large effects in the familial form of the disease to genes of smaller effect that may contribute to the sporadic form of ALS, which accounts for 90% of the patient population (1). On a cellular level, there has been a similar expansion in our understanding of the various pathogenic mechanisms that may lead to the selective killing of motor neurons in ALS. Leading hypotheses to date have centered on defective protein clearance and RNA processing, oxidative damage, inflammation, mitochondrial dysfunction, excitotoxicity, faulty axonal transport, and inadequate growth factor signaling (reviewed by Ferraiuolo and colleagues) (2). Neuronal death likely arises from a combination of these factors, which are likely responsible for the death or aberrant functioning of non-neuronal cells (i.e., astrocytes, microglia) that have been noted in various stages of the disease.

Relatively less attention has been paid, however, to the mechanisms underlying the triggering and progression of disease. Over twenty years ago, Eisen proposed that ALS originated from the upper motor neuron, and then spread via an anterograde "dying forward" process (3), whereas Chou and Norris stressed the primacy of the lower motor neuron and postulated a retrograde, or dying back, process (4). Brooks likened the inter-neuronal spread of ALS to the spread of polio virus and observed directionality, being weighted towards caudal progression. He longitudinally tracked symptom accrual in ALS and postulated that disease progression was consistent with axonal transport of an etiological agent (5). More recently, Ravits et al. observed spatially contiguous areas of motor neuron involvement, and hypothesized that motor neuron degeneration was fundamentally a focal process that spreads contiguously through the upper and lower motor neurons (6,7). Additionally, they also noted directionality, with the outward spread being weighted towards caudal progression. Further, they suggested that directionality of spread involves a number of factors, including variability in motor neuron susceptibility, differences in axonal length, dendritic arborization, and microenvironmental factors.

Finally, a number of other investigators have hypothesized that ALS, as well as other neurodegenerative illnesses, bear striking similarities to prion diseases. This line of thought contends that there is region-to-region spread of misfolded and aggregated proteins, much as self-replicating proteins accrue and disseminate in a prion disease such as Creutzfeldt-Jakob disease. Neurodegeneration, in this view, is in essence a proteinopathy, in which a variet

lile the

riety of

both normal and mutated proteins can behave as "seeds" or nucleation points fueling the misfolding of native proteins that are ultimately transmitted to contiguous cells. While there is clear similarity to prion diseases, "prions" and "prion-like" properties of proteins are distinct: "prions" are infectious, whereas "prion-like" proteins are not infectious, but have folding properties that induce misfolding.

This notion gained considerable credence in both in vitro and in vivo studies examining the aggregation and propagation of Tau and a-synuclein (8-10). Luk et al. (11), for example, demonstrated that the intracerebral injection of synthetic a-synuclein fibrils was sufficient to initiate the formation of Parkinson's-like Lewy bodies in mice. From this, they inferred a direct cell-to-cell transmission of a-synuclein, most likely affecting cells within interconnected neuronal pathways. By analogy, in ALS, several authors have suggested that misfolded proteins such as SOD1, FUS, and TDP-43 may also cause aggregation of their native counterparts via a prion-like mechanism (12-16).

Based on their view of the pathologic primacy of pTDP-43 cytoplasmic inclusions in sporadic ALS, Braak et al.(15) concluded that the locus of the initial insult was at the level of the cerebral cortex. And the nidus for spread resided in the neocortical pyramidal cells that are characterized by the length of their axons. Lesions in subcortical regions, in this scenario, stem from cell-to-cell transport of pTDP-43 pathology via anterograde axonal transport. The notion of a cerebral origin for ALS has been suggested by others, based on MEG imaging studies (17).

Commenting on disease progression in ALS, Grad and Cashman (13) invoked macropinocytosis as the mechanism of spread. They noted that both mutant and wild-type misfolded SOD1 can be secreted into the extracellular space via exosomes, small 30-80 nm-wide membrane-bound vesicles that normally contain and transport proteins and nucleic acids between cells. Exosomes have also been implicated in viral spread, as well as the spread of pathogenic

proteins in ALS, Alzheimer's, and Parkinson's disease (18-20). In addition to the exosomal pathway, Bowen et al. have demonstrated that a wide range of neurons exhibit phagocytic properties both in vitro and in vivo, including the ingestion of macromolecular debris from the death of one neuron by an adjacent neuron (21).

Although the body of research reviewed above has focused on a spreading of ALS amongst physically contiguous neurons, be it anterograde, retrograde, or via other mechanisms involving direct cell-to-cell contact, it incompletely explains certain clinical and in vitro data. Considering this, we propose that a critical factor involved in both the cause and spreading of ALS resides within the cerebrospinal fluid (CSF). While CSF as a route of disease spread has not been examined in neurodegenerative diseases, it is an accepted method of metastatic spread for cancer cells throughout the CNS (reviewed by Weston and colleagues) (22).

In this report, we review the key components of CSF production, including the role of the choroid plexus (CP) responsible for secreting it. Further we note the role of the CP in secreting proteins such as transthyretin that may be of interest in ALS (23,24), and comment on the CP's interface with the immune system. We then provide several lines of evidence from both clinical and cell culture research which support a potential role for CSF in the spread of ALS, and that suggest directions for future translational research.

Function, Circulation, and Anatomical Distribution of

Cerebrospinal Fluid

ventricl parench

The cerebrospinal fluid is in intimate contact with most regions of the central nervous system, circulating through four ventricles and the central canal of the spinal cord before flowing into the subarachnoid space (see Figure 1). Ependymal cells lining the interior surface of the freely permit diffusion of macromolecules within the interstitial fluid and brain renchyma. Periventricular sites accessible to the CSF include a variety of anatomical structures, e.g., the caudate, hippocampus, pineal gland, dentate gyrus, hypothalamus, periaqueductal gray matter of the mid-brain, and the pons-medulla. Additionally, there is a CSF "microcirculation" that originates at the pial surface and carries molecules into brain tissue via transport through the perivascular (Virchow-Robin) spaces of penetrating blood vessels (25,26).

In normal adults, total CSF volume is approximately 125-150 ml, and the entire volume is renewed about 3-4 times daily. Radiolabeled tracers injected intraventricularly are swept within minutes to regions distant from the injection site (27), convectively transported by pulsatile waves generated by arterial blood flow as well as currents produced by cilia on epithelial cells and pressure gradients created by the production and absorption of CSF.

ie CSF irculati ucts fr

provides buoyant support and a physical cushion for the brain's structures, but its circulation and turnover rate also make it a central player in both the removal of metabolic by-products from the brain, as well as the homeostatic regulation of a variety of solutes, including nutritive, trophic, and neuroendocrine factors.

The CSF is ultimately absorbed into the vasculature through arachnoid villi that are contiguous with the subarachnoid space overlying the surface of the brain and projecting into the main venous drainage of the CNS. The hydrostatic pressure of CSF is higher than the venous pressure, so the arachnoid villi essentially act as one-way valves returning the CSF to the blood stream. Since there isn't a traditional lymphatic system in the brain, the CSF serves as a surrogate, clearing metabolites secreted into the interstitial space surrounding neurons and glial cells. Molecules and drugs delivered into the subarachnoid space can be transported via the CSF microcirculation and delivered to neurons throughout the brain and spinal cord, making it a suitable approach for drug delivery in a variety of conditions (28).

Choroid Plexus and the Blood-CSF Barrier

Cerebrospinal fluid is produced primarily by the choroid plexus (29) (see Figure 1), structures that are essentially extensions of the ependymal cell layer of the ventricular wall. The outer layer of the CP is composed of cuboidal epithelial cells. This is the secretory machinery of the CP, and these cells rest on a basal lamina that in turn surrounds an inner core of connective and vascular tissue. Macrophages, leukocytes, dendritic cells, and fibroblasts populate the connective tissue stroma of the CP. The vascular bed at the CP's core provides a blood flow up to ten times greater than that found in the rest of the brain's vasculature in order to fuel the prolific activity of the CP epithelial cells. The CP plays a central role in controlling the extracellular environment of the brain. It secretes nutrients and essential proteins such as growth

factors and transthyretin (see Table 1), surveys the chemical and immunological status of the brain, and participates in CNS immune surveillance and repair processes following trauma.

In essence, the central nervous system (CNS) employs two circulations that work in tandem to maintain homeostasis and move nutrients in, and metabolic by-products out of the brain. The vascular system supplies the brain intrahemispherically via cerebral capillaries. The endothelial cells in these capillaries are joined by tight junctions that maintain the blood brain barrier (BBB). The tight junctions between the epithelial cells of the choroid plexuses are the basis of the blood cerebrospinal fluid barrier (BCSFB), since the endothelial cells within the CP vasculature do not possess tight junctions. The vascular and CSF circulations thus both play roles in secretion as well as reabsorption of solutes from the brain parenchyma.

Historically, the BBB has received the lion's share of pharmacologic, toxicologic, and pathologic attention. However, the BCSFB and the choroid plexuses are as critical as the BBB in these regards, and it could be argued that the CSF actually has a more intimate relationship with the brain than does the blood. With a broad array of transport systems, receptors, and the ability to synthesize many biologically active compounds (Table 1), the choroid plexus is ideally situated to distribute molecules both locally and globally within the brain (38). By means of convective distribution within the spinal fluid and diffusion across permeable membranes (ependyma and pia mater), bioactive molecules and solutes secreted by the CP gain widespread distribution throughout the brain, including the white matter (39).

Neurons are highly sensitive to both toxicants and pathogens, and the CP is strategically positioned to monitor as well as defend the brain against noxious molecules and potentially damaging cellular invasion. It possesses a complex, multi-layered detoxification system, with a full complement of Phase I-III enzymes for metabolizing drugs and other xenobiotics, organic ion transport systems, multidrug resistance proteins, and protective enzymes like glutathione-s-transferase and superoxide dismutase (40). It helps orchestrate the recovery process from trauma and disease, through secretion of a wide array of neuroprotective and trophic factors (e.g., glial cell line-derived neurotrophic factor, neurotrophins 3 and 4, basic-FGF, TGF-a and -P, IGF-2, transferrin, etc.) (30,40). The CP also serves as a site for neurogenesis, possibly due to its proximity to the subventricular zone (SVZ) (39,40).

Extensive invaginations on the basolateral sides of the CP epithelial cells combined with lush microvilli on the apical side provide a huge surface area for molecular transport, and it is believed that the total area for transport by the CP in the four ventricles is on the same order of

magnitude as the entire vasculature supplying the brain (41). The high metabolic demand of

choroid, required to transfer up to 75% of the water molecules diffusing from the plasma

sma int

CNS, is supported by an augmented blood flow.

)f the to the

Choroid Plexus/Immune System Interactions

The location and anatomy of the CP also position it to serve as a unique neuroimmunological interface, integrating signals emanating from the CNS parenchyma with incoming signals from circulating immune cells. In effect, the CP is a critical component of the intrinsic surveillance system of the CNS, responding to blood-borne pathogens and antigens in the CSF, as well as facilitating the resolution of inflammation by permitting the recruitment of inflammation-resolving leukocytes into the CNS. The CP functions as an entry gate for leukocytes and M2 monocytes into the CNS after injury via IFN-y signaling (42). This may be important in ALS, because microglial activation and T cell infiltration are pathological hallmarks of the disease. It has been hypothesized that this inflammatory response is not simply a late sequela of motor neuron degeneration, but is actively involving in tipping the scales between neuroprotection and neurodegeneration (reviewed by Henkel and colleagues) (43).

While the brain has traditionally been considered an immunologically privileged site, without lymph nodes or an extensive lymphatic system, in fact it is constantly being surveyed by immune cells circulating within the CSF (44), and just recently, the existence of functional lymphatic vessels draining the dural sinuses has been reported (45). The lack of tight junctions in the choroidal capillaries allows immune cells to migrate from the CP into the spinal fluid. Unlike the vasculature of the BBB, where immune cell trafficking mainly occurs in a variety of pathological conditions such as autoimmunity and ischemic insult, the capillaries of the CP constitutively express adhesion molecules and chemokines to permit the ongoing trafficking of regulatory leukocytes into the cerebrospinal fluid. The immune cell composition in the CP stroma and ventricular CSF differs from that of the blood, being dominated by memory CD4+ T cells. Under physiological conditions, the cytokine composition of the CSF (IL-13 and TGF-^2)

is largely immunosuppressive, a common feature of other immune-privileged sites like the eye and testes (46).

Resident microglial cells also inventory the brain parenchyma, can be rapidly activated, and assume a pro-inflammatory status in response to infection or tissue injury (47). In the past, the inflammatory response accompanying neurodegeneration was thought to be incidental. More recently, however, microglial and T cell activation have been demonstrated to play an important role in the rate of disease progression. Work in animal models of ALS provides compelling evidence for T cell-mediated down-regulation of the microglial inflammatory response (48).

The T cells resident in the CP and circulating in the CSF also recruit monocytes from the peripheral circulation into the brain, which can similarly down-regulate inflammation in the CNS (49-51). Therefore the CP plays an important neuroprotective role by reducing inflammation within the CNS during injury or disease (reviewed in Schwartz and Baruch) (52). In short, the resolution of inflammation in the brain is likely an active process involving both the CP and the CSF pathway, and it could be argued that dysregulation emanating from the CP contributes to the pathophysiology of neurodegenerative diseases.

CSF and CP in the Aging Brain

There are a number of changes in both the CSF and the CP that occur with aging that could be relevant to the development of ALS, a disease that primarily affects older adults. Firstly, it is well known that CSF secretion decreases with age (by up to 45% in some animal models) (53). CSF turnover is also reduced, going from a total renewal capacity of 3-4 times daily in young adults to less than twice daily in the elderly (54). CSF turnover rate is also known to decrease during a variety of neurodegenerative diseases. Absorption of CSF via the arachnoid villi overlying the surface of the brain diminishes with age, and the flow of spinal fluid in the spinal canal may be altered as a result of spondylitic changes (55). The functions of the CP are also highly energy-dependent, and with advancing age, the secretory ability of the CP's epithelial cells can decline. The height of these cells decreases about 10% throughout life (56), and infiltrating blood vessels in the CP also become thicker and more fragmented (57).

As CP secretory ability and CSF turnover rate decline, there is a concomitant reduction in the distribution of nutritive and reparative substances to the brain parenchyma and the clearance of noxious substances and metabolic by-products from the brain. This can lead to increased cellular stress and altered cell regenerative capacity, affecting neurogenesis in the SVZ and dentate gyrus (58). Relevant to this observation, Baruch et al.(59) identified the CP as the site of transcriptional changes that were linked to cognitive decline in senescent mice. This study identified a type I interferon-dependent gene expression profile that was also found in aged human brains. The IFN-I transcriptional response was induced by signals present in the CSF and it was found that blocking the IFN-I signaling could partially restore cognitive function and hippocampal neurogenesis in mice.

tore cognitive f

Also noteworthy is a recent report from Xie and colleagues, who proposed a key role for sleep in the clearance of metabolites from the brain (60). They demonstrated that during waking hours, CSF flow is restricted to the brain's surface, but it extends much deeper into the brain parenchyma during both sleep and anesthesia. The flow of CSF through the interstitial space was found to be reduced during waking to only 5% of that which occurs during sleep. Further, they found radiolabeled P-amyloid, a peptide that accumulates in the brain during waking and that has been implicated in the progression of Alzheimer's disease, was cleared twice as fast from the brains of mice who were asleep than awake. Other metabolites could similarly be cleared faster during sleep than during wakefulness.

The Puzzle of Selective Involvement of the Motor Neuron in ALS

One of the most salient features of ALS is its dominant impact on motor neurons, especially those in the brain stem and spinal cord. This begs the question of why these neurons are preferentially targeted during the disease. Some have argued that the long axonal processes of motor neurons make them susceptible to a variety of cellular pathologies such as defects in axonal transport. In humans, motor neuron axons can extend more than a meter in length, making these cells among the most unique in nature. Alterations in axonal structure are well documented in both ALS patients (61,62), and in transgenic mice bearing mutations in the SOD1 gene (6365). These axonal alterations include the inappropriate accumulation of neurofilaments, the most abundant structural components of large myelinated axons. Neurofilament accumulations are, in

fact, a pathological hallmark of both the sporadic and familial forms of the disease (66). The lower availability of protective heat shock proteins may also contribute to the selective vulnerability of motor neurons in ALS (67-70).

Primacy of CSF: Evidence from Clinical and In Vitro Studies

Could it be that the most straightforward explanation for the vulnerability of motor neurons in ALS is that they bear the brunt of the pathologic process due to their proximity to the cerebrospinal fluid pathway? Motor nuclei in the ventral horn of the spinal cord, for example, lie within a few millimeters of CSF flowing in the subarachnoid space (see Figure 1), and motor neurons in the most lateral portion of the ventral horn (i.e., neurons lying closest to the subarachnoid space containing CSF), are generally the first to be affected in ALS (71). This is consistent with a mode of onset that is customarily distal, involving either the hands or the feet. Similarly, nuclei of the 5 th, 7 th, 9th, 10th, and 12th cranial

nerves are commonly involved in ALS (72), and these nuclei also lie in close proximity to the CSF. Of these nuclei, the 9th, 10th, and 12th cranials tend to be more severely impaired during ALS than are the 5th and 7th, and these nuclei are in closer proximity to the CSF than are the 5th and 7th cranial nuclei. Finally, it should be pointed out that in spite of their proximity to the CSF pathway, neurons enervating the extraocular muscles and the pelvic sphincter are relatively resistant to the disease process. This is, however, likely due to their lower expression of matrix metalloproteinase-9 (73).

Several recent studies have analyzed the spread of ALS into different regions of the CNS over time, and have essentially arrived at the same conclusion; namely, that ALS starts focally within a single region and then spreads locally, by extending into contiguous neurons (6,74-78). Accordingly, the appearance of muscle weakness and atrophy in one limb, for example, would be expected to be followed by the appearance of similar findings in contiguous body regions rather than elsewhere. However, symptom spread from a lower limb to the brainstem is not readily accounted for by this contiguous neuron propagation hypothesis. And similarly, spread from musculature innervated by the bulbar region of the brain to a lower limb would also seem to require an alternate explanation, considering the lack of proximity of these neurons to one another.

Specifically, as shown in Table 2, 21-29% of patients in two of the above-referenced studies who had bulbar onset ALS next acquired involvement in their lower limbs. Likewise, the expected progression of the disease for patients with lower limb onset did not always follow the expected course, i.e., progression from one limb to the other. On follow-up, 14-15% of these patients were found to have difficulties with speech and/or swallowing.

îse [subseq

These results are not explained by the assumption of a single focal "seed" and subsequent simple linear cell-to-cell propagation. We propose instead that the initial seeding of ALS is mediated through the spinal fluid, and instances involving multi-centric onset are be st explained by spreading through the CSF rather than by axonal transmission. This may better fit a variety of observed clinical data and offer a more complete explanation for the progression of ALS.

Consistent with the above, a recent study by Sekiguchi et al.(79) suggested that there may, in fact, be multiple, focal sites of initiation associated with local spread of disease. Using EMG, this group demonstrated a noncontiguous pattern of involvement in many ALS patients. In short, multiple sites of disease initiation suggest seeding from a single source, i.e., the spinal fluid. In support of this, both antibodies (80) and oligonucleotides (81) are known to be robustly taken up by neurons in the brain stem and spinal cord following intraventricular injection (see Figure 2). Similarly, one can assume that other macromolecules, some of them toxic, could be taken up by neurons and glial cells from the CSF.

Along this line, Kaspar and his colleagues have convincingly demonstrated that astrocytes can secrete factors toxic to motor neurons (82). Astrocytes derived from neural progenitor cells taken from ALS patients selectively killed mouse motor neurons in a co-culture model of disease. Noteworthy was the finding that conditioned media derived from these astrocytes selectively upregulated a set of 22 genes encompassing chemokines, proinflammatory cytokines, and components of the complement cascade. And even more important may be the finding that motor neuron toxicity could be rescued when SOD1 expression was knocked out using a short hairpin RNA.

This is perhaps the best evidence to date suggesting that a protein such as wildtype or mutant SOD1 could be injurious to motor neurons. Since SOD1 is a constituent of the CSF (83), it is reasonable to conclude that an aggregated wildtype or mutant SOD1 molecule could be

seeded throughout the nervous system via the CSF pathway. Once taken up by neurons or glial cells, propagation could subsequently occur by axonal transport or contiguous spread through the extracellular space. Noteworthy is the finding that CSF obtained from ALS patients induces degeneration of cultured motor neurons (84). Equally important may be the report from Bi colleagues, who demonstrated that reactive astrocytes secrete LCN2 that is selectively tox

y toxic

neurons and that this protein can also be measured in the CSF of ALS patients (85).

Implications and Conclusion

and c to

Despite a huge body of research into genetics, epidemiology, diet, lifestyle, and the many mechanisms of cellular pathogenesis observable in ALS, a single unifying theory for the cause of the disease is lacking. In the midst of a seemingly dizzying array of complexity, could there be a simpler explanation that has eluded us?

If there is a unitary hypothesis for the cause of sporadic ALS, it would need to account for a number of factors. Among these are 1) A predominant involvement of motor neurons; 2) A uniform, worldwide distribution of the disease (with the notable exception of several foci of ALS occurring in the western Pacific); 3) Late age of disease onset; and 4) Its predilection for males.

If anatomy is destiny in the instance of ALS, the CSF and CP would take center stage as key players in seeding, and in some instances, propagating, the disease process. As previously reviewed, the intimate contact of CSF with motor neurons, its complex fluid dynamics during sleep, the critical role of the CSF and CP in CNS immune surveillance, and the extensive and rapid distribution from CSF of both toxic and reparative factors place it at a key nexus as a potential pathogenic factor in ALS.

The notion that neuronal spread in ALS is initiated by factors circulating in the cerebrospinal fluid has implications for research that could provide insight into both the cause and treatment of ALS. Firstly, it suggests that studying the composition of CSF could help identify the cause(s) of sporadic ALS. Secondly, it suggests that an effort to study the sources of potentially toxic molecules that find their way into the CSF may shed further light on the origins and spread of the disease. Thirdly, it suggests that research directed towards studying the role of the choroid plexus in ALS may be a fruitful avenue of inquiry. To our knowledge, this is a

neglected area of research. And finally, conceptionalizing sporadic ALS as a disorder of the CSF pathway suggests new therapeutic opportunities for this, and potentially for other, neurodegenerative diseases.


Dr. Joanne E. Martin, Professor of Pathology at the Blizard Institute of Queen Mary University of London, contributed critical commentary to the manuscript for which we are grateful.

Dr. Smith receives grant support from the Amyotrophic Lateral Sclerosis Association and has a commercial relationship with Isis Pharmaceuticals.

Dr. Bowser is a recipient of grants from NIH/NINDS and receives funding from Iron Horse Diagnostics, Inc., and from Knopp Biosciences, LLC.

Dr. Myers has nothing to disclose. Dr. Ravits has nothing to disclose.


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Figures Figure 1

Figure 2 2A

Table 1: A Sampling of Compounds Synthesized by the Choroid Plexus

Adrenomedulin Aquaporin-1

Apolipoprotein J/clusterin

Basic fibroblast growth factor-1 and -2

ß-Amyloid precursor protein

Brain-derived neurotrophic factor


Cystatin C


Glial cell line-derived neurotrophic factor Hepatocyte growth factor Insulin-like growth factor-2 Insulin-like growth factor binding proteins 2-6 Interleukin-1 ß Interleukin-6

Multidrug resistance-1 P glycoprotein Nerve growth factor Neurotrophin-3 and -4 Prostaglandin D synthase Retinoic acid Transferrin

Transforming growth factor-a Transforming growth factor-ß1, -ß2, -ß3 Transthyretin Tumor necrosis factor-a Vascular endothelial growth factor Vasopressin

Table 2: Progression of ALS into Non-Contiguous Neurons

Progression Fujimura - Kiyono et al. (74) McKenery et al.(75) >

Bulbar Lumbar 29% 21%

Lumbar Bulbar 14% 15%

Legends of Figures and Tables Figure 1 Key anatomical features of the cerebrospinal fluid pathway

The cerebrospinal fluid communicates with broad regions of the central nervous system, filling two lateral ventricles (A), the 3rd ventricle (B), and the 4th ventricle (C) in the brain's interior. CSF eventually flows from the ventricles into the subarachnoid space and its cisterns, and from there into sulci overlying the cortical surface, as well as the spinal cord's central canal (D). There is also a CSF "microcirculation" which originates at the pial surface and carries molecules into brain tissue via transport through the perivascular, or Virchow-Robin spaces, of penetrating blood vessels.

CSF is primarily secreted by the choroid plexus (E), a structure that is essentially an extension of the ependymal cell layer of the ventricular wall. The tight junctions between the epithelial cells of the choroid plexus are the basis of the blood cerebrospinal fluid barrier (BCSFB), since the endothelial cells within the CP vasculature itself do not possess tight junctions. Endothelial cells in cerebral capillaries, however, are joined by tight junctions that constitute the blood brain barrier (BBB).

The 5 th, 7 th, 9th, 10th, and 12th cranial nerve nuclei are frequently involved in ALS, as of course are motor neurons in the spinal cord. The proximity of the 5th, 7th, 10th, and 12th nerves to the CSF is illustrated. F, 5th cranial (trigeminal) nerve nucleus; G,7th cranial (facial) nerve nucleus; H, 12th cranial (hypoglossal) nucleus; I, dorsal motor nucleus of 10th cranial nerve (vagus); J, motor neurons of the anterior horn of the spinal cord. The relative proximity of spinal motor neurons to surrounding spinal fluid in the subarachnoid space (K) as well as in the central canal (D) is shown.

Figure 2 Uptake of intra-ventricularly injected macromolecules into neurons

2A) Immunofluorescent microscopy depicting the presence of antibody within spinal neurons two weeks after their intracerebroventricular infusion in mice. Anti-choline acetyltransferase (ChAT) (red) was used to label motor neurons in tissue sections obtained from the lumbar spinal cord, and a fluorescently conjugated rabbit anti-mouse secondary antibody (green) was used to detect the infused primary antibody. Adapted with permission from GrosLouis et al. (80).

2B) A chemically-modified 20-mer antisense oligonucleotide was infused into the right lateral ventricle of a normal rat for 14 days, after which tissues were collected and localization of the oligonucleotide was determined by immunostaining with an antibody that specifically recognizes the oligonucleotide. Immunostaining was noted at all levels of the spinal cord, with prominent uptake in the ventral horn. The arrow in the panel highlights uptake by a neuronal cell. Oligonucleotide was also found to be distributed to non-neuronal cells, including astrocytes and microglia, and was shown to be localized in brain parenchyma relevant to neurodegenerative diseases, including the hippocampus, substantia nigra, pons, and cerebellum (81).

and broadly distributed tl 'al axis. These include cytokines, growth, regulatory,

and neurotrophic factors, cell matrix proteins, proteases and protease inhibitors, binding and transport proteins, peptides, vitamins, and nucleosides.

A wide variety of

synthesized by the choroid plexus are secreted into the CSF

Table 1

Table adapted from: (30-37)

Table 2

Fujimura-Kiyono et al. (74) followed 150 patients with sporadic ALS and tracked the spread of symptoms from the initial area(s) involved to the appearance of symptoms at a secondary site. Patients were followed at 3-month intervals and assessed using the revised version of the ALS Functional Rating Scale. McKenery et al. (75) also followed 150 ALS patients, excluding patients who had multiple sites of disease onset, and classified symptoms as involving either cervical, lumbar, bulbar, or respiratory areas of the CNS. The percentage of patients in both studies having either a bulbar-to-lumbar spread of disease symptoms or a lumbar-to-bulbar spread is listed.