Subventricular Zone Under the Neuroinflammatory Stress and Parkinson’s Disease

Abstract

This review summarizes the effects of neuroinflammatory stress on the subventricular zone (SVZ), where new neurons are constitutively produced in the adult brain, especially focusing on the relation with Parkinson’s disease (PD), because the SVZ is under the control of dopaminergic afferents from the substantia nigra (SN). In Lewy bodies-positive-PD, microglia is known to phagocytoze aggregated α-synuclein, resulting in the release of inflammatory cytokines. The neurogenesis in the SVZ should be affected in PD brain by the neuroinflammatory process. The administration of lipopolysaccaharide is available as an alternative model for microglia-induced loss of dopaminergic neurons and also the impairment of stem cell maintenance. Therefore, the research on the neuroinflammatory process in the SVZ gives us a hint to prevent the outbreak of PD or at least slow the disease process.

Introduction

Brain plasticity in the adult was originally conceived as changes at the level of synaptic transmission, synaptic contacts, and gene expression. However, it became a more complicated concept since the continuous generation of neural stem cells in the adult had been reported. Altman and colleagues discovered a constitutive production of new neurons in the hippocampal dentate gyrus (Altman and Das 1965) and in the subventricular zone (SVZ) which is subadjacent to the ependyma lining the lateral ventricles (Altman 1969); these findings turned over the long-held consensus that the mammalian brain is a post-mitotic structure incapable of generating new neurons.

The adult SVZ with its unique cytoorganization retains many features of embryonic neurogenic niches (Doetsch et al. 1997; Alvarez-Buylla and Lim 2004), because adult neurogenesis consists of the following steps: (1) cell division of a stem cell resulting in one daughter stem cell and one with the potential to develop into a neuron, and (2) the newly generated neuroblast migrates to its final and appropriate destination in the brain. Therefore, the SVZ is composed of three major neurogenic cell types (Alvarez-Buylla et al. 2001; Alvarez-Buylla and García-Verdugo 2002). A specialized type of glial fibrillary acidic protein (+) astrocytes (termed B-cells) act as stem cells, because they have the potential to self-renew and to give rise to astrocytes, oligodendrocytes, and neurones (Doetsch et al. 1999). B-cells generate frequently dividing transit-amplifying C-cells (TACs), which can be identified by their expression of the epidermal growth factor receptor (EGFR) (Doetsch et al. 2002; Höglinger et al. 2004). Asymmetric division of C-cells gives rise to polysialic neural cell adhesion molecule (+)-restricted neural precursors (neuroblasts termed as A-cells). A-cells are destined to migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they are fate-committed to replace dead or dying granule cells in the OB (Lois and Alvarez-Buylla 1994; Jankovski and Sotelo 1996; Doetsch et al. 1999; Alvarez-Buylla and García-Verdugo 2002; Gritti et al. 2002; Ming and Song 2005) and to become GABAergic granule or periglomerular interneurons in the OB (Luskin 1993; Alvarez-Buylla and Lim 2004). Bulbar neurogenesis is highly involved in odor memory, because the balance between neurogenesis and apoptosis in the OB and RMS is influenced by olfactory sensory inputs (Gheusi et al. 2000; Rochefort et al. 2002; Enwere et al. 2004). Therefore, the constant supply of newly generated neural cells to the adult OB as neural turnover is prerequisite for the learning and memory in the sensation of smell. These facts indicate that the disruption of endogenous neurogenesis contributes to neurologic deficits and hinders recovery from neurodegeneration (Krathwohl and Kaiser 2004a, b; Kaul 2008; Peng et al. 2008; Waldau and Shetty 2008). The factors to govern the generation, migration, differentiation, integration, and survival of new neuroblasts in the SVZ include diffusible molecules such as neurotransmitters (Alvarez-Buylla 1997; Alvarez-Buylla et al. 2001; Alvarez-Buylla and Lim 2004; Hagg 2005; Lledo and Saghatelyan 2005). Therefore, it is essential to understand which specific receptor subtypes for neurotransmitters, hormones, and growth factors are expressed by each subset of the cells constituting the SVZ.

Collectively, it is to be expected that an alteration in brain neurotransmitter levels in the neurodegenerative diseases would affect adult neurogenesis in the SVZ with yet unknown functional consequences.

The SVZ–RMS–OB Pathway and Catecholaminergic Neurons

The locus coeruleus (LC) is considered to be a crucial site in the CNS for the response to stress. In fact, elevated norepinephrine metabolism in the LC due to peripheral lipopolysaccharide (LPS) administration as a model of inflammatory stress has been reported (Molina-Holgado and Guaza 1996; Lacosta et al. 1999; Hayley et al. 2001; Kaneko et al. 2001). Studies on rats showed that approximately 40% of the LC neurons project to the main OB and that noradrenergic fibers terminate densely in specific layers in the OB (Shipley et al. 1985; McLean et al. 1989). Probes for α_1A/D- and α_2C-adrenergic receptor mRNAs used in in situ hybridization histochemistry react strongly with specific neurons and layers, respectively, in the OB (Nicholas et al. 1993; Pieribone et al. 1994). In addition, prominent immunoreactivity indicating α_2A-adrenergic receptors is observed in the OB (mainly localized in the granular cell layer) (Talley et al. 1996).

The projection of noradrenergic neurons into the SVZ has been poorly understood. Instead, dopamine receptors in the SVZ have been precisely mapped by using immunohistochemical and electron microscopy studies. Dopamine receptors are classified into either D1-like (D1 and D5) or D2-like (D2, D3, and D4) ones according to structural and functional homology. They possess a highly homologous transmembrane domain and share the intracellular cascades. However, the second messenger cascades utilized by these two subfamilies are distinctively different. The D1-like receptors activate the Gα_s/olf family of G proteins resulting in stimulating cAMP production by adenylate cyclase (AC); The D2-like ones activate the Gα_i/o family of G proteins leading to the inhibition of cAMP production by AC (Beaulieu and Gainetdinov 2011). Neither D1 nor D2 receptor was found in B-cells (Höglinger et al. 2004). On the contrary, TACs identified as highly proliferative EGFR (+) cells in the adult SVZ are embedded in a rich network of dopaminergic afferents that form synapse-like structures (Höglinger et al. 2004). While D1-like receptors are found in the cytoplasm but not the cell membrane of TACs, D2-like receptors are most abundantly expressed in TAC cell membranes and sparsely expressed in SVZ astrocyte cell membranes (Höglinger et al. 2004). Höglinger et al. (2004) also showed that dopamine stimulates the proliferation of EGFR (+) cells through D2 receptors. TACs also express D3 receptors, whereas neuroblasts and SVZ astrocytes do not (Kim et al. 2010). A-cells express both D1- and D2-like receptors (Coronas et al. 2004; Höglinger et al. 2004; Winner et al. 2006). mRNA expression and in vivo studies have implicated the expression of D3 receptor along with the embryonic and adult SVZ neurogenesis (Diaz et al. 1997; Van Kampen et al. 2004; Kim et al. 2010). The more striking finding is the fact that the dopaminergic projections to the SVZ from the substantia nigra (SN) have been demonstrated in rats (Höglinger et al. 2004; Winner et al. 2006), mice (Baker et al. 2004), and non-human primates (Freundlieb et al. 2006). In addition, Freundlieb et al. demonstrated that the dopaminergic fibers in the SVZ in the primates originate, at least in part, in the pars compacta of the SN (Freundlieb et al. 2006). Post-mortem studies in humans have identified dopaminergic fibers in contact with EGFR (+) cells in the SVZ, which are presumably TACs (Höglinger et al. 2004). These anatomical observations support the existence of a nigro-subventricular dopaminergic projection terminating on TACs, thus raising the hints of its effect on function. It should be noted that the neurotransmitter dopamine contributes to the ontogenesis of the mammalian brain by regulating neural precursor cell proliferation. Dopamine and its receptors appear early during embryonic development in the highly proliferative germinal zones of the brain (Voorn et al. 1988; Lidow and Rakic 1995; Diaz et al. 1997; Ohtani et al. 2003).

SVZ and Parkinson’s Disease

Parkinson’s disease (PD) is one of the neurodegenerative diseases characterized by death of dopaminergic neurons in the SN (Goedert 2001; Kuhn et al. 2006; Benner et al. 2008; Reynolds et al. 2008; Glass et al. 2010) and, although not in each case examined, the formation of inclusions known as Lewy bodies (LBs) (Spillantini et al. 1997, 1998a; Wakabayashi et al. 1997; Goedert 1999; Spillantini and Goedert 2000; Takeda et al. 2000; Goedert 2001; Trojanowski and Lee 2003; Lundvig et al. 2005). LBs, which primarily contain aggregated α-synuclein, are pathological markers of a group of diseases collectively categorized as “α-synucleinopathies” (Wakabayashi et al. 1997; Spillantini et al. 1998a, b; Goedert 1999; Spillantini and Goedert 2000; Takeda et al. 2000).

During a chronic and slowly progressive process in PD, neuronal dysfunction at the level of synaptic transmission, synaptic contacts, and axonal and dendritic degeneration occur. In addition, numbers of functional neurons in neurogenic regions, and adult neurogenesis are also altered or decreased, i.e., chronic neurodegeneration in PD affects stem cell maintenance, proliferation, survival, and functional integration (Höglinger et al. 2004). Prominent clinical features of PD are motor symptoms such as bradykinesia, tremor, rigidity and postural instability, and non-motor-related PD symptoms (olfactory deficits, autonomic dysfunction, depression, cognitive deficits, and sleep disorders). Non-dopamine brain regions affected in PD have recently attracted increasing interest, because the onsets of the non-motor symptoms such as olfactory dysfunction are observed early in the course of the disease (Korten and Meulstee 1980; Hawkes et al. 1997; Tolosa and Poewe 2009) and because a subset of these functions is connected to the stem and progenitor cell populations in the hippocampus and SVZ–RMS–OB axis. In the PD-model animals which are deprived of their dopaminergic neurons in the SN, SVZ proliferation is decreased. These observations indicate that PD is associated with decreased neurogenesis (Baker et al. 2004; Höglinger et al. 2004; Winner et al. 2006; O’Keeffe et al. 2009; Cova et al. 2010). The overexpression of human wild-type α-synuclein in transgenic mice has a negative impact on adult neurogenesis, i.e., decreased number in neuroblasts and newly generated neurons in the SVZ–RMS–OB systems (Winner et al. 2004, 2008). Post-mortem studies of patients with PD have demonstrated a decreased number of proliferative cells in the SVZ (Höglinger et al. 2004).

PD and Neuroinflammation

The cause of sporadic PD is unknown, with uncertainty about the role of environmental toxins and genetic factors (Moore et al. 2005; Farrer 2006). Theoretically, the progressive neurodegeneration of PD can be produced by the chronic exposure to neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Langston et al. 1983), 6-hydroxydopamine (Ungerstedt 1968), paraquat (Liou et al. 1997), and rotenone (Betarbet et al. 2000). MPTP blocks the mitochondrial electron transport chain by inhibiting complex I (Nicklas et al. 1985) and the abnormalities in complex I activity are identified in PD (Greenamyre et al. 2001), which causes the energy failure (i.e., ATP depletion) in the cells and exposes them to oxidative stress. Mitochondrial DNA mutations have not yet been identified in PD patients. A growing body of evidence suggests that the accumulation of misfolded abnormal proteins directly induced by protein malconformations (as believed to be the case with α-synuclein) or due to the damage to the cellular machinery to detect and degrade misfolded proteins (as believed to be the case with Parkin) is likely to be a key event in PD neurodegeneration. The toxic PD model and the genetic implication in inherited forms of PD comprise two major hypotheses regarding the pathogenesis of PD. However, they are not mutually exclusive, because the demise of dopaminergic neurons in SN is determined by the points where these two routes come across in their own cascades. For example, oxidative damage to α-synuclein can enhance its ability to misfold and aggregate is one example of such an interaction (Giasson et al. 2000).

On the other hand, until two decades ago, the CNS was considered an immunologically privileged site not normally accessed by circulating immune cells in the absence of inflammation or injury. Dendritic cells with specialized antigen-presenting capabilities are not present under normal conditions, but when microglia sense danger through Toll-like receptor 4 (Tlr4), they secrete inflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, reactive oxygen species (ROS), and nitric oxide (NO) to act on astrocytes and induce secondary inflammatory responses (Hirsch and Hunot 2009; Saijo et al. 2009).

α-Synuclein is a highly soluble natively unfolded protein expressed throughout the CNS. It is physiologically present in the presynaptic terminals of neurons (Maroteaux et al. 1988; Clayton and George 1999). As mentioned earlier, in sporadic PD and in most forms of familial PD, the accumulation and the aggregation of α-synuclein from monomers can result in the formation of intermediate state oligomers, which lead to neuronal cell death (Polymeropoulos et al. 1997; Krüger et al. 1998; Zarranz et al. 2004; Danzer et al. 2007; Lee 2008; Roodveldt et al. 2008). Therefore, it can be argued that α-synuclein is implicated in a series of complex pathological processes that cause the death of dopaminergic neurons, regardless of the form of PD (Corti et al. 2011). Extracellular α-synuclein released from the dead cells is phagocytosed by microglia (Zhang et al. 2005), and the aggregated, nitrated, and oxidized forms of α-synuclein induce microglia activation followed by activation of NADPH oxidase and production of ROS (Zhang et al. 2005; Reynolds et al. 2008). BV-2 cells (a microglia cell line) endocytose α-synuclein via lipid rafts by GM1 of cell surface gangliosides in vitro (Park et al. 2009). α-Synuclein released from neuronal cells are also readily endocytosed by astrocytes (Lee et al. 2010). These findings suggest that α-synuclein-mediated neurotoxicity is enhanced by microglia and/or astrocytes activation and release of pro-inflammatory cytokines. Therefore, when neurons are under protein conformational stresses forming α-synuclein aggregates, secreted forms of α-synuclein potentially act as a messenger between neurons and glia, inducing neuroinflammatory responses.

Microglia activation and an increase in astrocytes are observed in PD (Boka et al. 1994; Mogi et al. 1994, 2000; Hirsch et al. 1998; Gao et al. 2002). Reactive microglia along with LBs are found in the SN of PD patients (McGeer et al. 1988). In addition, increased levels of cytokines in the cerebrospinal fluid and in the blood have been reported (Nagatsu and Sawada 2005). An increase in the number of astrocytes (Damier et al. 1993) and dystrophic astrocytes (Braak et al. 2007) are detected in the post-mortem PD brains. Positron emission tomography also suggests increased glial activation in PD patients (Gerhard et al. 2006). Although Nurr1, an orphan nuclear receptor binding to target genes as a monomer, homodimer, or heterodimer with retinoid X receptors (Maira et al. 1999; Aarnisalo et al. 2002; Wang et al. 2003), is required for the generation and maintenance of dopaminergic neurons (Zetterström et al. 1997) with rare mutations associated with familial PD (Le et al. 2003), Nurr1 also functions as an activator or a repressor of cell type-specific responses in non-neuronal cells to inflammatory stimuli (Barish et al. 2005; Pei et al. 2005; Doi et al. 1997). Reduction in Nurr1 levels exacerbates inflammatory responses in microglia and astrocytes, leading to degeneration of tyrosine hydroxylase (TH)-positive neurons, suggesting that Nurr1 protects dopaminergic neurons by restraining the activity of microglia and astrocytes (Saijo et al. 2009). Therefore, inflammation is a common secondary devastating mechanism in PD. Collectively, PD is a complex disorder that not only involves death of dopaminergic neurons, but also provokes widespread inflammation in the brain (Block and Hong 2007; McGeer and McGeer 2008).

In order to explain the etiology of all forms of PD based on an inflammatory process, the “α-synucleinopathy” encounters the following problems; (1) LBs are intraneuronal inclusions, (2) extracellular α-synuclein has not been reported in PD brain, (3) neither humans nor animals develop LB from MPTP administration, and (4) Parkin-linked autosomal recessive early onset PD is devoid of LB formation. Despite a lack of LBs in MPTP Parkinsonism, activated microglia and dopaminergic neuronal loss were still found in patients even 16 years after MPTP exposure and 18-year-old non-human primates given MPTP years earlier (McGeer and McGeer 2008). Therefore, MPTP administration can initiate inflammatory reaction in CNS that becomes self-sustaining after MPTP has disappeared. A recent report from Tran et al. (2011) is worthy of note. They reported that the mouse parkin gene promoter contains a nuclear factor-κB (NF-κB) regulatory site and that exposure to LPS or TNF induces a decrease in Parkin mRNA and protein in microglia, macrophages and neuronal cells blockable by inhibitors of NF-κB signaling. However, it is still inconclusive whether the pathophysiological process of Parkin-mutated PD converges to the neuroinflammatory activation.

SVZ, PD, Neuroinflammation, and LPS

This chapter deals with the reports from the viewpoint that inflammation can cause PD, although there are some controversies in the field and the interrelationships among many parameters are not fully worked out. For example, the experiments done in our laboratory (referred below) were performed according to the working hypothesis of inflammation-inducing PD.

LPS is a cell wall component of Gram-negative bacteria. LPS binds to a CD14, a glycosylphosphatidylinositol-linked membrane protein; and together with the extracellular adaptor protein MD-2, LPS binds to Tlr4 expressed by microglia (Beutler 2004). Tlr4 is the key transmembrane receptor for LPS effects, because mice with either a point or null mutation in the Tlr4 gene are insensitive to LPS (Rosenberg 2002; Palsson-McDermott and O’Neill 2004). Transduction through Tlr4 triggers a cascade of intracellular events that leads to the transcription of inflammatory and immune response genes and is responsible for most of the inflammatory events caused by bacterial infection because of its potency in causing a “cytokine storm.” Peripheral administration of LPS is thought to cause internally a strong toxic stress similar to that in infectious diseases (Breder et al. 1994; Quan et al. 1997a, b; Nadeau and Rivest 1999; Iwase et al. 2000; Rivest 2003; Tonelli et al. 2003), and a single systemic injection of LPS causes a robust increase in the expressions of various genes encoding pro-inflammatory cytokines and chemokines in microglia, as well as proteins of the complement system (Quan et al. 1997a, b; Lacroix et al. 1998; Nguyen et al. 2002; Chakravarty and Herkenham 2005; Bonow et al. 2009). In addition, as found on macrophages, LPS in the circulation activates Tlr4 on microglia in the circumventricular organs and/or choroid plexus, inducing TNF-α synthesis at the sites (Nadeau and Rivest 2000). Then, TNF-α activates the pro-inflammatory transcription factor NF-κB in adjacent microglia, resulting in pro-inflammatory gene transcription (including TNF-α itself) in these cells and progression of the inflammatory response in the parenchyma of the brain. Therefore, TNF-α plays a detrimental role in neural survival/differentiation (Monje et al. 2003; Liu et al. 2005).

The role of bacterial or viral infection as an initiating factor in human PD is unclear. However, intracranial infusion of bacterial LPS is available as a model for microglia-induced loss of TH-positive dopaminergic neurons in rodents (Castano et al. 1998; Kim et al. 2000), because the cascade following the binding of LPS with Tlr4 converges to the activation of non-neural cells (microglia and astrocytes). Activated microglia in inflammatory settings can inhibit neurogenesis (Ekdahl et al. 2003; Butovsky et al. 2006). Stereotaxic injection of LPS into SN of genetically engineered mice with human α-synuclein causes inflammatory reactions of glia with no differences among the genes introduced into the mice, whereas the loss of TH-positive neurons after LPS injection is more prominent in mice expressing mutated α-synuclein than the ones expressing wild-type (Gao et al. 2008). As mentioned earlier, LPS and TNF suppress Parkin expression via NF-κB (Tran et al. 2011). Systemic LPS administration into Parkin-null mice increases the vulnerability of nigral dopamine neurons to inflammation-related degeneration (Frank-Cannon et al. 2008). Systemic LPS administration into mice following exposure to MPTP induces more extensive neurodegeneration in the SN (Cardona et al. 2006). Tlr4 is abundantly expressed by neural stem/progenitor cells and LPS decreases the proliferation of cultured neural stem/progenitor cells via a NF-κB-dependent mechanism (Rolls et al. 2007). Systemically injected LPS mimicking the neuroinflammatory settings also increases the apoptotic cells in murine SVZ–RMS–OB axis (Mori et al. 2005, 2010). The absence of Tlr4 enhances proliferation and neuronal differentiation (Rolls et al. 2007). It should be noted that, in in vitro setting, Tlr4 directly modulates self-renewal and the cell-fate decision of neuronal progenitor cells (Rolls et al. 2007).

In addition, injection of LPS into the rodent brain increases the levels of inflammatory mediators such as cyclooxygenase-2 and inducible NO synthase prior to loss of dopaminergic neurons (Hunter et al. 2007). Indeed, mediators released by the immune cells, like cytokines and NO, negatively regulate adult neurogenesis (Vallières et al. 2002; Monje et al. 2003; Liu et al. 2005). However, because the duration of the effects initiated by LPS triggering has not been precisely defined to explain a long-lasting neurodegenerative process in PD, this point remains to be clarified to establish the concept of “inflammation-induced PD.” In the line of the evidences, recent report by Kaneko et al. (2009) is worthy of note. They found that LPS can extend the life-span of primary-cultured microglia for more than 2 months keeping them in active state.

The links among inflammation, oxidative stress, and PD are supported by an overwhelming number of studies that implicate inflammatory processes in the progressive loss of the nigral dopaminergic neurons and the increase in the apoptotic cell death in the SVZ which receives the nigral dopaminergic axons. However, it still remains to be determined whether anti-inflammatory therapy in humans has a beneficial effect in preventing or slowing the progression of PD (Tansey and Goldberg 2010).

Closing Remarks

At the end of this review, we would again emphasize the role of SVZ–RMS–OB axis. It is reasonable to search for the way to arrest the apoptotic cell death in the SVZ–RMS–OB axis caused by immune-associated mechanisms, because the arrest of apoptosis might become a valuable tool to prevent the outbreak of PD or at least to slow the disease process. In this respect, a series of reports from a group at the NIH is worthy of note. Benicky et al. (2011) reported that the systemic administration of candesartan, a centrally acting angiotensin II AT₁ receptor blocker (ARB), to normotensive rats decreased the acute brain inflammatory response to the administration of LPS and reduced microglia activation. These effects were widespread in the brain (not restricted to well-known brain target areas for circulating proinflammatory factors and LPS such as the hypothalamic paraventricular nucleus and the subfornical organ). Therefore, this ARB may offer a new therapeutic approach for the cessation of the inflammatory damage to the cells migrating in the SVZ–RMS–OB pathway.