Neurovascular Aspects of Amyotrophic Lateral Sclerosis

NEUROVASCULAR ASPECTS OF AMYOTROPHIC LATERAL SCLEROSIS

Maria Carolina O. Rodrigues1,6, Diana G. Hernandez-Ontiveros1, Michael K. Louis1, Alison E. Willing1,2,3,4, Cesario V. Borlongan1,2, Paul R. Sanberg1,2,4,5, Ju´lio C. Voltarelli6 and Svitlana Garbuzova-Davis1,2,3,4 1

Center of Excellence for Aging & Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, Florida, USA 2 Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, Florida, USA 3 Department of Molecular Pharmacology and Physiology, University of South Florida, Morsani College of Medicine, Tampa, Florida, USA 4 Department of Pathology and Cell Biology, University of South Florida, Morsani College of Medicine, Tampa, Florida, USA 5 Department of Psychiatry, University of South Florida, Morsani College of Medicine, Tampa, Florida, USA 6 Department of Internal Medicine, Ribeira ˜ o Preto School of Medicine, University of Sao Paulo, Sao Paulo, Brazil

Abstract I. Introduction II. BBB/BSCB Impairment in ALS A. Experimental Studies Evidencing BBB/BSCB Dysfunction B. Human Studies Evidencing BBB/BSCB Dysfunction III. Future Perspectives IV. Conclusion Acknowledgments References

Abstract

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with a complicated and poorly understood pathogenesis. Strong evidence indicates impairment of all neurovascular unit components including the blood–brain and blood– spinal cord barriers (BBB/BSCB) in both patients and animal models. The present review provides an updated analysis of the microvascular pathology and impaired BBB/BSCB in ALS. Based on experimental and clinical ALS studies, the roles of cellular components, cell interactions, tight junctions, transport systems, cytokines, INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 102 DOI: 10.1016/B978-0-12-386986-9.00004-1

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Copyright 2012, Elsevier Inc. All rights reserved. 0074-7742/12 $35.00

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matrix metalloproteinases, and free radicals in the BBB/BSCB disruption are discussed. The impact of BBB/BSCB damage in ALS pathogenesis is a novel research topic, and this review will reveal some aspects of microvascular pathology involved in the disease and hopefully engender new therapeutic approaches.

I. Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with an estimated incidence of 1.6 in 100,000 people per year, and a reported prevalence of 4 per 100,000 (Hirtz et al., 2007). The disorder affects upper and lower motor neurons, leading to progressive muscle atrophy, paralysis, and death typically within 3–5 years from diagnosis (Haverkamp et al., 1995; Rowland and Shneider, 2001). Most ALS cases are sporadic (SALS) with only 5–10% genetically linked (FALS), and of those that have familial etiology, 20% show missense mutations in the Cu/Zn superoxide dismutase (SOD1) gene (Rosen et al., 1993). Numerous hypotheses exist regarding ALS pathogenesis, including glutamate excitotoxicity, oxidative stress, mitochondrial dysfunction, neurofilament accumulation, protein mishandling, altered glial function, viral infections, impaired trophic support, and immune imbalance (Bruijn et al., 2004; Consilvio et al., 2004; Deng et al., 2011; Mitchell and Borasio, 2007; Pasinelli and Brown, 2006; Rothstein, 2009; Saleh et al., 2009; Strong et al., 2005; Van Den Bosch et al., 2006), but the roles played by these deficiencies, as primary or cumulative motor neuron insults, still need to be determined. The blood–brain and blood–spinal cord barriers (BBB/BSCB) have a crucial role in regulating the exchange of molecules between the central nervous system (CNS) and the peripheral blood, and protecting the CNS from hazardous fluctuations in plasma composition (Abbott and Romero, 1996; Ballabh et al., 2004; Bradbury, 1985; Nag, 2003; Pardridge, 1999). Exchange by free diffusion is limited to molecules weighing less than 450 Da; substances with greater size require specific transporting mechanisms (Pardridge, 2005). Endothelial cells and their tight junctions are the main components of the BBB/BSCB system, while astrocyte end-feet, perivascular macrophages, pericytes, and the basement lamina also have integral roles. There is a dynamic interaction between environmental factors and endothelial and CNS resident cells, the basement membrane, and migrated immune cells (Dermietzel and Krause, 1991), continuously modulating the permeability and selectivity of the BBB/BSCB. Therefore, functional or structural impairment of any of the barrier components may impair this system protecting the CNS, thus endangering the cerebral homeostasis.

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Impairment of the BBB, BSCB, or blood–cerebrospinal fluid barrier (BCSFB) has been suggested in ALS (Garbuzova-Davis et al., 2007a,b, 2008, 2011; Nicaise et al., 2009a,b; Zhong et al., 2008). Although neuronal death seems to be a final event in ALS, and one event associated with most manifestations of the disease, dysfunction or structural damage of the BBB/BSCB or BCSFB may contribute to ALS pathogenesis. Initial reports of altered BCSFB permeability in ALS were published over 25 years ago (Annunziata and Volpi, 1985; Leonardi et al., 1984), and more recent research indicates impairment of the BBB and BSCB, both in animal models and in patients. Indeed, BBB/BSCB leakage is observed in SOD1 animal models of ALS since presymptomatic stage of disease, hence preceding neuronal death (Nicaise et al., 2009a; Zhong et al., 2008). These observations may change the focus of investigation in ALS: from a neuronal-centered to a broader, possibly endothelium-centered approach. Therefore, the classification of ALS as a neurovascular disease (Garbuzova-Davis et al., 2011) provides a basis for future therapeutic studies, investigations perhaps targeting BBB/BSCB repair.

II. BBB/BSCB Impairment in ALS

The first evidence of BCSFB impairment appeared in the 1980s: abnormal serum proteins and complement in the CSF of ALS patients (Annunziata and Volpi, 1985; Leonardi et al., 1984). These observations were followed by detection of blood-borne substances in the CNS tissue of ALS patients (Donnenfeld et al., 1984), suggesting BBB/BSCB leakage. However, follow-up studies (Bilic et al., 2006; Pirttila et al., 2004) did not confirm the initial findings. A few years later, the topic saw renewed interest, paralleling BBB investigations in other neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease. More recent studies not only evaluate the BCSFB but also focus on BBB/BSCB competence and have established that the neurovascular unit, composed of the CNS microvascular endothelium, pericytes, astrocyte end-feet, extracellular matrix, and neurons, might be impaired in ALS. Table I lists the evidence of BBB/BSCB impairment in ALS from animal and human studies. Figure 1 provides a schematic overview of known hallmarks of BBB/BSCB alterations in ALS compared to an intact, normally functioning neurovascular unit.

A. EXPERIMENTAL STUDIES EVIDENCING BBB/BSCB DYSFUNCTION In 2007, Garbuzova-Davis and colleagues (2007b) showed Evans blue leakage in spinal cord capillaries of G93A SOD1 mice at 13 weeks of age, indicating functional impairment of the BSCB in early stage disease. The study also

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Table I FAVORABLE AND OPPOSING EVIDENCE OF BBB/BSCB IMPAIRMENT IN ALS PATIENTS AND ALS ANIMAL MODELS. References Description of Evidence

Experimental

Human

Functional leakage of BBB/BSCB Garbuzova-Davis et al. (2007b) and Nicaise et al. (2009a) Altered blood flow or capillary Zhong et al. (2008) Arhart (2010) and Rule et al. lengths (2010) Microhemorrhages or Garbuzova-Davis et al. (2007a) Verstraete et al. (2010) perivascular hemosiderin and Zhong et al. (2008) (unsupporting) Endothelial cell degeneration or Garbuzova-Davis et al. (2007a,b) Henkel et al. (2009) damage and Nicaise et al. (2009a) Astrocyte-capillary dissociation Garbuzova-Davis et al. (2007a,b), Miyazaki et al. (2011) Nicaise et al. (2009b), and Miyazaki et al. (2011) Decreased basement membrane Garbuzova-Davis et al. (2007a,b) Miyazaki et al. (2011) components and Miyazaki et al. (2011) Altered MMP activity/expression Miyazaki et al. (2011), Fang et al. Beuche et al. (2000), Demestre (2010), and Soon et al. (2010) et al. (2005), and NiebrojDobosz et al. (2010) Downregulation of junctional Zhong et al. (2008) and Miyazaki Miyazaki et al. (2011) and et al. (2011) Henkel et al. (2009) complex proteins Altered endothelial transporter Garbuzova-Davis et al. (2007a,b) Niebroj-Dobosz et al. (2010) protein expression and Milane et al. (2010)

demonstrated endothelial damage through downregulation of the transporter protein Glut-1 and CD146 expressions, associated with decreased laminin content of the basement membrane in capillaries. These findings were later confirmed by a study on the same mouse model, showing ultrastructural alterations to the vessels surrounding degenerating neurons in the brainstem and spinal cord (cervical and lumbar), in both early and late stages of disease (Garbuzova-Davis et al., 2007a). Electron microscopy evaluations detected degenerated endothelial cells and astrocytes, mitochondrial degeneration within endothelial cells, extensive extracellular edema, and swelling of astrocyte end-feet adjacent to capillaries. Capillary rupture was also indicated by the presence of erythrocytes in the extracellular space of brainstem microvessels in early symptomatic G93A mice. Zhong et al. (2008) suggested that BSCB disruption precedes neurovascular inflammation and might initiate disease symptoms in G93A mice. Western blot analysis evidenced diminished levels of zonula occludens 1 (ZO-1), occludin, and claudin-5 tight junction proteins and Glut-1. The alterations were observed at the presymptomatic disease stage, while markers of endothelial activation

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TJ Tight junction AJ Adherens junctions

Vacuoles

MMP-2 & -9

Nutrients

Hemosiderin

Blood-borne pathogens

IgG

Adhesion receptor Cytokine receptors

Aquaporin-4

Cytokine (i.e., TNF, IL-1)

VEGF

Damaged BBB/BSCB in ALS

Intact BBB/BSCB

Reactive astrocyte

Astrocyte

Resting microglia Endothelial cell

Perivascular macrophage

Activated microglia Edema

TJ

Basement membrane AJ Pericyte

Astrocyte end-feet

Healthy motor neuron

Impaired motor neuron

FIG. 1. Overview of a normal and an ALS-impaired neurovascular unit: structural and functional levels. The neurovascular unit in the brain and spinal cord is composed of the microvascular endothelium, perivascular cells, astrocytes, neurons, and the extracellular matrix. Healthy CNS homeostasis depends on a normally functioning blood–brain/spinal cord barrier (BBB/BSCB), which separates the brain and spinal cord from the systemic blood circulation and regulates the exchange of various substances. The BBB/BSCB is a unique complex system composed of endothelial cells and their tight/adherens junctions, astrocyte end-feet, perivascular macrophages, pericytes, and the basement membrane. Integrity of all barrier elements is essential for optimal neuron function. In ALS, BBB/BSCB alterations at structural and functional levels have been noted in both patients and animal models. Endothelial cell and astrocyte end-feet degeneration, altered basement membrane composition, tight junction and transporter system impairment, serum protein leakage (i.e., IgG), hemosiderin deposits, extensive extracellular edema (increased aquaporin-4 expression), downregulation of VEGF expression, and MMP-2/MMP-9 activation are significant hallmarks of this BBB/BSCB impairment. The barrier damage allows entry of blood-borne harmful substances, entry which might accelerate motor neuron degeneration. Additionally, cytokines (i.e., TNF and IL-1) released from activated microglia and reactive astrocytes may detrimentally affect not only motor neurons but also the vascular endothelium. The numerous indications of diminished integrity of the BBB/BSCB, a key component of the neurovascular unit, strongly point to microvascular impairment as a central feature in ALS pathogenesis.

(intercellular adhesion molecule 1, ICAM-1) and inflammation (monocyte chemoattractant protein-1, MCP-1) and cycloxygenase-2 (COX-2) were yet undetected. Still prior to motor neuron loss and inflammatory changes, the investigators also detected 10–15% reductions in total capillary length and 30–45%

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decreases in expected blood flow in the spinal cords of SOD1 transgenic mice. These findings indicate a possible role for hypoxia and/or ischemia in provoking inflammation and degeneration in ALS. In addition, microhemorrhages and hemosiderin deposits were found in spinal cord parenchyma, demonstrating BSCB functional impairment and disruption. Miyazaki et al. (2011) also evaluated BSCB integrity in G93A SOD1 mice, supporting previous findings. The authors observed progressive downregulation of occludin and platelet-endothelium cell adhesion molecule (PECAM-1 or CD31) and perivascular collagen IV, associated with increased activity of the gelatinase matrix metalloproteinase 9 (MMP-9), indicating endothelial cell and basement membrane involvement in microvascular pathological changes. Dissociation between astrocyte end-feet and capillary vessels reinforced the findings of structural damage of the BSCB. All of these observations preceded motor neuron death, agreeing with Zhong et al. (2008). The alterations were mainly detected in the ventral horns of the spinal cords, areas most affected by ALS. Surprisingly, the quantification of collagen IV in gray matter tissue evidenced upregulation of the protein, opposing immunohistochemical observations in the perivascular areas. The divergent results were ascribed to increased glial production of collagen IV, as a consequence of disease progression and neuroinflammation. The literature, however, presents some conflicting reports. A study on the G93A SOD1 rat model of ALS demonstrated ultrastructural alterations of the capillaries only at symptomatic stage (Nicaise et al., 2009a). Perivascular swollen astrocyte end-feet, Evans blue leakage, reduced mRNA expression of ZO-1 and occludin, and of agrin, a basement membrane component, were observed in animals at symptomatic, but not presymptomatic age. Conversely, IgG and hemosiderin deposits, indicators of BBB/BSCB leakage, were detected in the brainstem and lumbar spinal cord at presymptomatic stage. As a novel observation in ALS, the authors showed increased expression of aquaporin-4 (AQP4) mRNA and protein in the gray matter of end-stage SOD1 rats (Nicaise et al., 2009b). Electron microscope and immunohistochemical analyses demonstrated that the edematous and degenerated perivascular astrocytic end-feet contained high concentrations of AQP4, suggesting that the AQP channels may contribute to intra- and extracellular edema. Milane et al. (2010) demonstrated increased P-glycoprotein (P-gp) expression in brain capillaries of a G86R SOD1 mouse model of ALS. Along with the breast cancer resistance protein, P-gp transports undesired substances, such as excess glutamate, neurotoxins, and ions from the CNS to the blood, and thus has a protective role (Banks, 2008). It is hypothesized that P-gp upregulation may be a consequence of glutamate excitotoxicity and upregulation of proapoptotic genes (caspase, FAS, TNF receptor), cytokines, and enzymes (NO synthase and COX-2) in the activated glia, besides oxidative stress induced by the SOD1 mutation. When evaluated in the colon and jejunum, P-gp expression was similar in control

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and ALS mice, indicating that increased expression was CNS specific and related to the neurodegenerative process (Milane et al., 2010). Additional reports suggest vascular endothelial growth factor (VEGF) as a neuroprotective agent, possibly related to hypoxia and angiogenesis. During the past decades, it has been shown that VEGF is progressively involved in the pathogenesis of ALS as well as in the pathogenesis of other neurodegenerative diseases (Oosthuyse et al., 2001; Wang et al., 2007). VEGF is associated with motor neuron survival, in addition to its known role in vasculogenesis and angiogenesis (Storkebaum et al., 2004). Additionally, a concordance between VEGF and ALS was established when Vegfd/d mice, which do not upregulate VEGF as a response to hypoxia, presented similar neurological presentation and progressive motor neuron death as the G93A SOD1 transgenic mouse model of ALS (Oosthuyse et al., 2001). Moreover, the addition of VEGF to motor neuron cultures from Vegfd/d mice improved motor neuron survival. These findings suggest that, under a hypoxic environment, neural tissue fails to produce adequate amounts of VEGF, exposing motor neurons to death. Furthermore, crossbreeding of G93A SOD1 mutant mice with mice overexpressing VEGF resulted in animals with delayed motor neuron loss and disease progression versus G93A mice (Wang et al., 2007). Furthermore, VEGF levels in CSF of ALS patients significantly increase with disease duration, and importantly, chronic hypoxia has been implicated in ALS pathology (Iłzecka, 2004). A pathway through hypoxic states has been described, with increased VEGF production by endothelial cells, inducing phosphorylation of the ZO-1 junctional protein and thereby decreasing endothelial tightness (Fischer et al., 2004). This pathway includes participation of the protein apelin, a ligand for the GPCR protein, encoded by the Apj gene. Apelin is regulated by hypoxia and, with VEGF, helps regulate vascular development and endothelial cell proliferation (Kunduzova et al., 2008). Kasai et al. (2011) recently evaluated the role of apelin in ALS. Double mutant apelin knockout/G93A SOD1 mice presented accelerated disease progression and reduced survival when compared to SOD1 mutant mice alone. Moreover, evaluations of hippocampal cell cultures exposed to the proapoptotic agent hydrogen peroxide evidenced increased motor neuron survival when apelin plus VEGF, but not apelin alone, were added to the cultures. This evidence, although still preliminary, indicates the importance of VEGF in ALS and suggests a relationship between vasculature, hypoxia, and motor neuron survival.

B. HUMAN STUDIES EVIDENCING BBB/BSCB DYSFUNCTION Inflammation and immune cell activation have been detected in the central nervous tissue of ALS patients and are associated with motor neuron death (Boille´e et al., 2006; Donnenfeld et al., 1984; Engelhardt and Appel, 1990;

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Engelhardt et al., 1993, 1995; Henkel et al., 2004). Early studies with human tissue from ALS patients found IgG and complement (C3 and C4) deposits in the spinal cord and motor cortex, suggesting BBB/BSCB disruption (Donnenfeld et al., 1984). Engelhardt and Appel (1990) also detected active macrophages and IgG within the endoplasmic reticulum of motor neurons in ALS patients. The observation was complemented by a second study from the same group, showing that IgG from sera of ALS patients induced death of a motor neuron cell line (VSC 4.1) in vitro (Engelhardt et al., 1995). More recently, Henkel et al. (2009) demonstrated diminished mRNA expression of occludin and ZO-1 in human lumbar spinal cord tissue from both sporadic and familial forms of ALS. These results agreed with the experimental findings, confirming loss of endothelial integrity, and indicating BSCB disruption that might contribute to disease pathogenesis. Garbuzova-Davis et al. (2010) reported that the numbers of circulating endothelial cells were reduced in the peripheral blood of ALS patients with moderate or severe disease. Circulating endothelial cells are considered markers for endothelial damage (Blann et al., 2005), and their numbers are increased in several vascular diseases, such as acute myocardial infarct and acute ischemic stroke (Chong et al., 2006; Nadar et al., 2005), as well as in disorders in which the endothelium is secondarily involved, such as traumatic brain injury and ovarian cancer (Gong et al., 2011; Su et al., 2010). A possible explanation for the unexpected results in ALS would be a lack of endothelial shedding, resulting in the attachment of new ECs over the damaged cells, resulting in a multilayer endothelium (Garbuzova-Davis et al., 2010). Indeed, electron microscopy images of ALS mouse tissue have revealed multiple layers of endothelial cells in spinal cord capillaries (Garbuzova-Davis et al., 2007a). Also, deficient MMP or excessive protease inhibitor (TIMP) activities may be involved with this event, since endothelial shedding results from MMP activity degrading basement membrane components (Shapiro et al., 2010; Taraboletti et al., 2002). MMPs have been enrolled in the pathogenesis of ALS by several studies. Ono et al. (1998a) observed that patients with long-term ALS had fewer skin bedsores than would be expected for their neurologic impairment and bed confinement. As a possible mechanism, excess MMP activity would lead to degradation of collagen IV from the epithelial extracellular matrix, therefore reducing the adherence between epidermis and dermis. The increased mobility of the skin would prevent the formation of bedsores. In a different publication from the same authors (Ono et al., 1998b), decreased perivascular collagen densities in the ventral horns and posterior lateral funiculi of human spinal cords, correlating degradation of the basement membrane with areas of neurodegeneration in ALS, were reported. Other studies detected high levels of MMP-2 and MMP-9 in the CNS tissue, serum, and CSF of ALS patients and animal models (Beuche et al., 2000; Demestre et al., 2005; Fang et al., 2010; Niebroj-Dobosz et al., 2010). Bossolasco et al. (2010) recently detected abnormal MMP and TIMP productions by

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mesenchymal cells from the bone marrow of ALS patients, suggesting that ALS pathology is not restricted to the CNS. Other authors support this hypothesis, correlating skin manifestations observed in diseased humans and mice with excess MMP-9 activity (Fang et al., 2009; Soon et al., 2010). More recently, increased levels of laminin and hyaluronic acid, both components of the basement membrane, were observed in the skin of ALS patients, possibly associated, through still undefined mechanisms, with the degradation of perivascular collagen IV (Ono et al., 2000a,b). Oxidative stress is also known as an important pathological determinant in ALS (Cookson and Shaw, 1999; Robberecht, 2000), which might directly affect BBB/BSCB integrity. Reactive oxygen species (ROS) are a major product of mitochondrial activity in neural cells. In physiological conditions, specific endogenous antioxidants such as the superoxide dismutase and the glutathione peroxidase are sufficient to scavenge ROS. In pathological conditions, however, oxidative stress is enhanced by inflammation, excitotoxicity, mitochondrial dysfunction, and microglial activation. In consequence, excessive ROS reacts with proteins from the endothelial cell membranes, such as the ATPase, affecting the transcellular transport pathway (Pun et al., 2009). According to Yamauchi et al. (2007), endothelial cell cultures exposed to nitric oxide (NO) reduced activity of the P-gp efflux pump. Additionally, oxidative stress may impair the function of tight junction proteins, through phosphorylation and structural alterations. Hydrogen peroxide affects the distribution of occludin and ZO-1 along the endothelial cell membrane, while peroxynitrite decreases endothelial expression of claudin-5 (Kar et al., 2010). Oxidative stress may still induce activation of MMPs, especially the gelatinases MMP-2 and MMP-9 which, once activated, directly affect the BBB/BSCB, degrading tight junction proteins and components of the endothelial basement membrane (Kar et al., 2010; Nakashima et al., 2002; Rosenberg et al., 1998). However, not all the evidence points to BBB/BSCB disruption in ALS, Verstraete et al. (2010) published a study in which magnetic resonance imaging (MRI) evaluations of SALS patients did not detect microvascular bleeding. These results conflict with the pathological hemosiderin deposits observed in nervous tissue from ALS animal models. The MRI evaluations were limited to the brain, excluding the spinal cord, where ALS microbleeds are usually more pronounced in mice (Nicaise et al., 2009a; Zhong et al., 2008) and might be more expected in humans. Additionally, the hemorrhagic manifestations observed in mouse models of ALS may be related to the SOD1 mutation and, thus, absent from the disease pathogenesis of the sporadic form. Finally, it is hypothesized that ALS may be caused by chronic cerebrospinal vascular insufficiency (Arhart, 2010), an idea which is complementary to that of chronic hypoxia in the nervous tissue of ALS patients (Iłzecka, 2004). A series of publications from an Italian group (Zamboni et al., 2009a,b,c) described

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pathological venous constrictions impairing cerebral and spinal drainages, which were reversed by percutaneous luminal angioplasties. Although limited to multiple sclerosis patients, the investigators demonstrated disease remission and functional improvement after the procedure (Zamboni et al., 2009c). Moreover, the report is interesting for the discussion about how the high venous pressure generated by the venous reflux distends the capillaries and mechanically separates the intercellular tight junctions, initiating BBB/BSCB leakage and subsequently damaging the CNS. Other studies corroborate the idea that hemodynamic alterations may alter permeability of the BBB/BSCB (Collins et al., 2006; Krizanac-Bengez et al., 2006) and support the blood flow alterations observed by Zhong et al. (2008), preceding inflammation and motor neuron injury. Additional reinforcement comes from Rule et al. (2010), who observed a correlation between reduced capillary blood flow in brains of ALS patients and disease severity.

III. Future Perspectives

Today, it is known that, beyond being a purely motor neuron disease, ALS involves deleterious influences from an inflammatory and toxic environment, reinforced by the participation of the peripheral immune system, all of which contribute to motor neuron death (Rothstein, 2009). Inflammation is a key element in the process of motor neuron degeneration, involving participation of activated microglia and astrocytes, T lymphocytes, IgG, and numerous cytokines observed in the brainstem and spinal cord in both ALS patients and animal models (Alexianu et al., 2001; Boille´e et al., 2006; Consilvio et al., 2004; Donnenfeld et al., 1984; Engelhardt and Appel, 1990; Engelhardt et al., 1993,1995; Hall et al., 1998; Henkel et al., 2004; McGeer and McGeer, 2002). Inflammation may precede motor neuron injury and may also initiate BBB/BSCB damage, impairing endothelial cell function. In fact, Mantovani et al. (1992) stated that the inflammatory environment in ALS affected endothelial cell gene expression, altering cell function. However, the exact role of inflammation upon the endothelial cell alteration observed in ALS still needs to be determined. Further investigations are required to expand initial findings on microvascular pathology in ALS, including the BBB/BSCB alterations, since barrier damage seems to play an important, yet incompletely deciphered, role in ALS pathogenesis. Patients with familial ALS carrying the SOD1 mutation and the transgenic rodent models expressing mutant SOD1 have greatly contributed to the understanding of ALS pathogenesis. However, pathological mechanisms should be established without the involvement of the misfolding mutant SOD1 protein, in

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order to clarify the pathogenesis of SALS cases. Structural and functional barrier analyses in SALS patients are essential for this investigation. Our ongoing study analyzing brain and spinal cord postmortem tissues from ALS patients should contribute to a better understanding of these mechanisms.

IV. Conclusion

There is compelling evidence that the neurovascular unit (Hawkins and Davis, 2005; Vangilder et al., 2011) is impaired in both patients and animal models of ALS. Functional leakage of proteins, increased cell migration to the CNS, impairment of tight junction and transporter systems, activation of metalloproteinase, altered basement membrane composition, degeneration of endothelial cell and astrocyte end-feet and, more recently, blood flow alterations contribute to the idea of microvascular impairment as a central feature in ALS pathogenesis. The concept, also associated with other CNS diseases such as Alzheimer’s disease (Bell and Zlokovic, 2009), stroke ( Jung et al., 2010), and multiple sclerosis (Vos et al., 2005), greatly improves our knowledge of ALS pathogenesis. Since the BBB/BSCB is part of the tightly integrated neurovascular unit, barrier repair may promote neuron survival and lead to new therapeutic approaches for ALS.

Acknowledgments

This work was supported in part by the Muscular Dystrophy Association (Grant #92452) and the USF Department of Neurosurgery and Brain Repair.

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