A preferential dysfunction/loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) accounts for the main motor symptoms of Parkinson’s disease (PD), the most common degenerative movement disorder. However, the neuronal loss is not stochastic, but rather displays regionally selectivity, indicating the existence of different DA subpopulations in the SNpc. To identify the underlying molecular determinants is thereby instrumental in understanding the pathophysiological mechanisms of PD-related neuron dysfunction/loss and offering new therapeutic targets. Recently, we have demonstrated that aldehyde dehydrogenase 1 (ALDH1A1) is one such molecular determinant that defines and protects an SNpc DA neuron subpopulation preferentially affected in PD. In this review, we provide further analysis and discussion on the roles of ALDH1A1 in the function and survival of SNpc DA neurons in both rodent and human brains. We also explore the feasibility of ALDH1A1 as a potential biomarker and therapeutic target for PD.
Parkinson’s disease; Substantia nigra pars compacta; Dopaminergic neuron; Aldehyde dehydrogenase 1; α-synuclein; Neurodegeneration; Aging
The substitution of Proline with Serine at residue 56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) has been linked to an atypical autosomal dominant form of familial amyotrophic lateral sclerosis 8 (ALS8). To investigate the pathogenic mechanism of P56S VAPB in ALS, we generated transgenic (Tg) mice that heterologously express human wild-type (WT) and P56S VAPB under the control of a pan-neuronal promoter Thy1.2. While WT VAPB Tg mice did not exhibit any overt motor behavioral phenotypes, P56S VAPB Tg mice developed progressive hyperactivities and other motor abnormalities. VAPB protein was accumulated as large punctate in the soma and proximal dendrites of both corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs) in P56S VAPB Tg mice. Concomitantly, a significant increase of endoplasmic reticulum stress and unfolded protein response and the resulting up-regulation of pro-apoptotic factor CCAAT/enhancer-binding protein homologous protein expression were observed in the CSMNs and SMNs of P56S VAPB Tg mice. However, only a progressive loss of CSMNs but not SMNs was found in P56S VAPB Tg mice. In SMNs, P56S VAPB promoted a rather selective translocation of VAPB protein onto the postsynaptic site of C-boutons that altered the morphology of C-boutons and impaired the spontaneous rhythmic discharges of SMNs. Therefore, these findings provide new pathophysiological mechanisms of P56S VAPB that differentially affect the function and survival of CSMNs and SMNs in ALS8.
Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA) neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs) in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI) GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.
Chronic neuroinflammation is thought to play an etiological role in Alzheimer’s disease (AD), which is characterized pathologically by amyloid and tau formation, as well as neuritic dystrophy and synaptic degeneration. The causal relationship between these pathological events is a topic of ongoing research and discussion. Recent data from transgenic AD models point to a tight spatiotemporal link between neuritic and amyloid pathology, with the obligatory enzyme for β-amyloid (Aβ) production, namely β-secretase-1 (BACE1), is overexpressed in axon terminals undergoing dystrophic change. However, the axonal pathology inherent with BACE1 elevation seen in transgenic AD mice may be secondary to increased soluble Aβ in these genetically modified animals. Here we explored the occurrence of the AD-like axonal and dendritic pathology in adult rat brain affected by LPS-induced chronic neuroinflammation. Unilateral intracerebral LPS injection induced prominent inflammatory response in glial cells in the ipsilateral cortex and hippocampal formation. BACE1 protein levels were elevated the ipsilateral hippocampal lysates in the LPS treated animals relative to controls. BACE1 immunoreactive dystrophic axons appeared in the LPS-treated ipsilateral cortex and hippocampal formation, colocalizing with increased β-amyloid precursor protein and Aβ antibody (4G8) immunolabeling. Quantitative Golgi studies revealed reduction of dendritic branching points and spine density on cortical layer III and hippocampal CA3 pyramidal neurons in the LPS-treated ipsilateral cerebrum. These findings suggest that Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration occur in wildtype mammalian brain in partnership with neuroinflammation following LPS injection.
Amyloid Pathogenesis; Neuritic Dystrophy; Neurodegeneration; Neuroplasticity; Synaptic Pathology
Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). Here we show that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIβ (PKARIIβ). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged LRRK2-null mice after treatment with a Drd1 agonist. Notably, a Parkinson’s disease-related LRRK2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIβ also induced excessive PKA activity in the SPNs. Our findings reveal a new regulatory role of LRRK2 in PKA signaling, and provide a new pathogenic mechanism of SPN dysfunction in Parkinson’s disease.
Leucine-rich repeat kinase 2 (LRRK2), implicated in familial Parkinson’s disease (PD), was recently identified as a major susceptibility gene for Crohn’s disease (CD) by genome-wide association studies (GWAS). We found that LRRK2 deficiency confers enhanced susceptibility to experimental colitis in mice. Mechanistic studies showed that LRRK2 is a potent negative regulator of NFAT and a component of a previously described RNA-protein complex involving a non-coding RNA repressor of NFAT (NRON). Colitis in LRRK2 deficient mice is exacerbated by enhanced NFAT1 nuclear localization. Moreover, the risk-associated allele Met2397 identified in CD GWAS causes reduced LRRK2 protein expression, which, in light of our unexpected observation that LRRK2 is a negative regulator of NFAT, suggests a pathological mechanism important in human disease.
The spinal cord is composed of distinct neuronal groups with well-defined anatomic connections. In some transgenic models of Alzheimer’s disease (AD) amyloid plaques develop in this structure, although the underlying cellular mechanism remains elusive. We attempted to explore the origin, evolution and modulation of spinal β-amyloid (Aβ) deposition using transgenic mice harboring five familiar AD-related mutations (5XFAD) as an experiential model. Dystrophic neuritic elements with enhanced β-secretase-1 (BACE1) immunoreactivity (IR) appeared as early as 2 months of age, and increased with age up to 12 months examined in this study, mostly over the ventral horn (VH). Extracellular Aβ IR emerged and developed during this same period, site-specifically co-existing with BACE1-labeled neurites often in the vicinity of large VH neurons that expressed the mutant human APP. The BACE1-labled neurites almost invariably colocalized with β-amyloid precursor protein (APP) and synaptophysin, and frequently with the vesicular glutamate transporter-1 (VGLUT). Reduced IR for the neuronal specific nuclear antigen (NeuN) occurred in the VH by 12 months of age. In 8 month-old animals surviving 6 months after an unilateral sciatic nerve transection, there were significant increases of Aβ, BACE1 and VGLUT IR in the VN of the ipsilateral relative to contralateral lumbar spinal segments. These results suggest that extracellular Aβ deposition in 5XFAD mouse spinal cord relates to a progressive and amyloidogenic synaptic pathology largely involving presynaptic axon terminals from projection neurons in the brain. Spinal neuritic plaque formation is enhanced after peripheral axotomy, suggesting a retrograde transneuronal modulation on pathogenesis.
Alzheimer’s disease; amyloidogenesis; BACE1; neuritic dystrophy; synaptoplasticity
Subpopulations of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) display a differential vulnerability to loss in Parkinson’s disease (PD); however, it is not clear why these subsets are preferentially selected in PD-associated neurodegeneration. In rodent SNpc, DA neurons can be divided into two subpopulations based on the expression of aldehyde dehydrogenase 1 (ALDH1A1). Here, we have shown that, in α-synuclein transgenic mice, a murine model of PD-related disease, DA neurodegeneration occurs mainly in a dorsomedial ALDH1A1-negative subpopulation that is also prone to cytotoxic aggregation of α-synuclein. Notably, the topographic ALDH1A1 pattern observed in α-synuclein transgenic mice was conserved in human SNpc. Postmortem evaluation of brains of patients with PD revealed a severe reduction of ALDH1A1 expression and neurodegeneration in the ventral ALDH1A1-positive DA subpopulations. ALDH1A1 expression was also suppressed in α-synuclein transgenic mice. Deletion of Aldh1a1 exacerbated α-synuclein–mediated DA neurodegeneration and α-synuclein aggregation, whereas Aldh1a1-null and control DA neurons were comparably susceptible to 1-methyl-4-phenylpyridinium–, glutamate-, or camptothecin-induced cell death. ALDH1A1 overexpression appeared to preferentially protect against α-synuclein–mediated DA neurodegeneration but did not rescue α-synuclein–induced loss of cortical neurons. Together, our findings suggest that ALDH1A1 protects subpopulations of SNpc DA neurons by preventing the accumulation of dopamine aldehyde intermediates and formation of cytotoxic α-synuclein oligomers.
Deposition of β-amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer’s disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although less is clear about the change of the enzyme’s activity in AD brain. Cerebrospinal fluid (CSF) Aβ peptides are considered to derive from brain parenchyma, thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored a possibility of Aβ production and secretion by the choroid plexus (CP). Specific binding density of [3H]-L-685,458, a radiolabeled high affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with comparable ages and postmortem delays. The CP in postmortem samples exhibited exceptionally high [3H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins but released Aβ40 and Aβ42 into the medium. These results suggest that γ-secretase activity appears not altered in the cerebrum in AD related to aged control, nor correlated with regional amyloid plaque pathology. The choroid plexus appears to represent a novel non-neuronal source in the brain that may contribute Aβ into cerebrospinal fluid, probably at reduced levels in AD.
β-amyloid; BACE1; γ-secretase; anti-Aβ therapy; AD biomarker
β-Secretase-1 (BACE1) is the rate-limiting enzyme for the genesis of amyloid-β (Aβ) peptides, the main constituents of the amyloid plaques in the brains of Alzheimer’s disease (AD) patients. BACE1 is being evaluated as an anti-Aβ target for AD therapy. Recent studies indicate that BACE1 elevation is associated with axonal and presynaptic pathology during plaque development. Evidence also points to a biological role for BACE1 in axonal outgrowth and synapse formation during development. Axonal, including presynaptic, pathology exists in AD as well as many other neurological disorders such as Parkinson’s disease, epilepsy, stroke, and trauma. In this review, we discuss pharmaceutical BACE1 inhibition as a therapeutic option for axonal pathogenesis, in addition to amyloid pathology. We first introduce the amyloidogenic processing of amyloid-β protein precursor and describe the normal expression pattern of the amyloidogenic proteins in the brain, with an emphasis on BACE1. We then address BACE1 elevation relative to amyloid plaque development, followed by updating recent understanding of a neurotrophic role of BACE1 in axon and synapse development. We further elaborate the occurrence of axonal pathology in some other neurological conditions. Finally, we propose pharmacological inhibition of excessive BACE1 activity as an option to mitigate early axonal pathology occurring in AD and other neurological disorders.
aging; Alzheimer’s disease; anti-amyloid therapy; dementia; dystrophic neurites; neurodegenerative disorders; neuroplasticity; senile plaques; synaptic dysfunction
Recent genome-wide association studies indicate that a simple alteration of Leucine-rich repeat kinase 2 (LRRK2) gene expression may contribute to the etiology of sporadic Parkinson's disease (PD). However, the expression and regulation of LRRK2 protein in the sporadic PD brains remain to be determined. Here, we found that the expression of LRRK2 protein was enhanced in the sporadic PD patients using the frontal cortex tissue from a set of 16 PD patients and 7 control samples. In contrast, no significant difference was detected in the level of LRRK2 mRNA expression between the control and PD cases, suggesting a potential post-transcriptional modification of the LRRK2 protein expression in the sporadic PD brains. Indeed, it was identified that microRNA-205 (miR-205) suppressed the expression of LRRK2 protein through a conserved-binding site at the 3′-untranslated region (UTR) of LRRK2 gene. Interestingly, miR-205 expression was significantly downregulated in the brains of patients with sporadic PD, showing the enhanced LRRK2 protein levels. Also, in vitro studies in the cell lines and primary neuron cultures further established the role of miR-205 in modulating the expression of LRRK2 protein. In addition, introduction of miR-205 prevented the neurite outgrowth defects in the neurons expressing a PD-related LRRK2 R1441G mutant. Together, these findings suggest that downregulation of miR-205 may contribute to the potential pathogenic elevation of LRRK2 protein in the brains of patients with sporadic PD, while overexpression of miR-205 may provide an applicable therapeutic strategy to suppress the abnormal upregulation of LRRK2 protein in PD.
Background: Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause Parkinson disease.
Results: LRRK2 binds directly to three β-tubulin isoforms at the luminal face of microtubules and suppresses α-tubulin acetylation. Interaction is weakened by the R1441G LRRK2 GTPase domain mutant.
Conclusion: LRRK2 modulates microtubule stability.
Significance: Deregulation of microtubule-dependent processes likely contribute to neurodegeneration in Parkinson disease.
Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.
Lrrk2; Microtubules; Molecular Genetics; Parkinson Disease; Tubulin; GTPase Mutation; RocCOR; Cytoskeletal Dynamics; Growth Cone; Tubulin Acetylation
Association studies have identified several signals at the LRRK2 locus for Parkinson's disease (PD), Crohn's disease (CD) and leprosy. However, little is known about the molecular mechanisms mediating these effects. To further characterize this locus, we fine-mapped the risk association in 5,802 PD and 5,556 controls using a dense genotyping array (ImmunoChip). Using samples from 134 post-mortem control adult human brains (UK Human Brain Expression Consortium), where up to ten brain regions were available per individual, we studied the regional variation, splicing and regulation of LRRK2. We found convincing evidence for a common variant PD association located outside of the LRRK2 protein coding region (rs117762348, A>G, P = 2.56×10−8, case/control MAF 0.083/0.074, odds ratio 0.86 for the minor allele with 95% confidence interval [0.80–0.91]). We show that mRNA expression levels are highest in cortical regions and lowest in cerebellum. We find an exon quantitative trait locus (QTL) in brain samples that localizes to exons 32–33 and investigate the molecular basis of this eQTL using RNA-Seq data in n = 8 brain samples. The genotype underlying this eQTL is in strong linkage disequilibrium with the CD associated non-synonymous SNP rs3761863 (M2397T). We found two additional QTLs in liver and monocyte samples but none of these explained the common variant PD association at rs117762348. Our results characterize the LRRK2 locus, and highlight the importance and difficulties of fine-mapping and integration of multiple datasets to delineate pathogenic variants and thus develop an understanding of disease mechanisms.
α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.
The brain is capable of remarkable synaptic reorganization following stress and injury, often using the same molecular machinery that governs neurodevelopment. This form of plasticity is crucial for restoring and maintaining network function. However, neurodegeneration and subsequent reorganization can also play a role in disease pathogenesis, as is seen in temporal lobe epilepsy and Alzheimer’s disease. β-Secretase-1 (BACE1) is a protease known for cleaving β-amyloid precursor protein into β-amyloid (Aβ), a major constituent in amyloid plaques. Emerging evidence suggests that BACE1 is also involved with synaptic plasticity and nerve regeneration. Here we examined whether BACE1 immunoreactivity (IR) was altered in pilocarpine-induced epileptic CD1 mice in a manner consistent with the synaptic reorganization seen during epileptogenesis. BACE1-IR increased in the CA3 mossy fiber field and dentate inner molecular layer in pilocarpine-induced epileptic mice, relative to controls (saline-treated mice and mice 24–48 h after pilocarpine-status), and paralleled aberrant expression of neuropeptide Y. Regionally increased BACE1-IR also occurred in neuropil in hippocampal area CA1 and in subregions of the amygdala and temporal cortex in epileptic mice, colocalizing with increased IR for growth associated protein 43 (GAP43) and polysialylated-neural cell adhesion molecule (PSA-NCAM), but reduced IR for microtubule-associated protein 2 (MAP2). These findings suggest that BACE1 is involved in aberrant limbic axonal sprouting in a model of temporal lobe epilepsy, warranting further investigation into the role of BACE1 in physiological vs. pathological neuronal plasticity.
Aberrant neuroplasticity; Temporal lobe epilepsy; Mossy fiber sprouting; Dystrophic neurites; Beta-secretase; Alzheimer’s disease
The comorbidity between epilepsy and Alzheimer's disease (AD) is a topic of growing interest. Senile plaques and tauopathy are found in epileptic human temporal lobe structures, and individuals with AD have an increased incidence of spontaneous seizures. However, why and how epilepsy is associated with enhanced AD-like pathology remains unknown. We have recently shown β-secretase-1 (BACE1) elevation associated with aberrant limbic axonal sprouting in epileptic CD1 mice. Here we sought to explore whether BACE1 upregulation affected the development of Alzheimer-type neuropathology in mice expressing mutant human APP, presenilin and tau proteins, the triple transgenic model of AD (3×Tg-AD). 3×Tg-AD mice were treated with pilocarpine or saline (i.p.) at 6–8 months of age. Immunoreactivity (IR) for BACE1, β-amyloid (Aβ) and phosphorylated tau (p-tau) was subsequently examined at 9, 11 or 14 months of age. Recurrent convulsive seizures, as well as mossy fiber sprouting and neuronal death in the hippocampus and limbic cortex, were observed in all epileptic mice. Neuritic plaques composed of BACE1-labeled swollen/sprouting axons and extracellular AβIR were seen in the hippocampal formation, amygdala and piriform cortices of 9 month-old epileptic, but not control, 3×Tg-AD mice. Densities of plaque-associated BACE1 and AβIR were elevated in epileptic versus control mice at 11 and 14 months of age. p-Tau IR was increased in dentate granule cells and mossy fibers in epileptic mice relative to controls at all time points examined. Thus, pilocarpine-induced chronic epilepsy was associated with accelerated and enhanced neuritic plaque formation and altered intraneuronal p-tau expression in temporal lobe structures in 3×Tg-AD mice, with these pathologies occurring in regions showing neuronal death and axonal dystrophy.
Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ) and IKK-related (IKKε and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.
Of the various genetic factors contributing to the pathogenesis of Parkinson’s disease (PD), only mutations in α-synuclein (α-syn) and LRRK2 genes cause clinical and neuropathological phenotypes closely resembling the sporadic cases. Therefore, studying the pathophysiological functions of these two PD-related genes is particularly informative in understanding the underlying molecular pathogenic mechanism of the disease. PD-related missense and multiplication mutations in α-syn may cause both early- and late-onset PD, whereas various PD-related LRRK2 missense mutations may contribute to the more common late-onset PD. While intensive studies have been carried out to elucidate the pathogenic properties of PD-related mutant α-syn and LRRK2, our knowledge of their normal functions and their potential genetic interplay remains rudimental. In this review, we summarize the progress made regarding the pathophysiological functions of α-syn, LRRK2 and their interaction in PD, based on the available literature and our unpublished observations.
14-3-3; α-synuclein; actin; autophagy; ER; Golgi apparatus; leucine-rich repeat kinase 2; Lewy body; microtubule; mitochondria; Parkinson’s disease; proteasome
Amyloid precursor protein (APP) has long been linked to the neurodegeneration of Alzheimer’s disease (AD), but the associated cell death has been difficult to capture in vivo, and the role of APP in effecting neuron loss is still unclear. Olfactory dysfunction is an early symptom of AD with amyloid pathology in the olfactory epithelium correlating well to the brain pathology of AD patients. As olfactory sensory neurons (OSNs) regenerate continuously with immature and mature OSNs co-existing in the same olfactory epithelium, we sought to utilize this unique system to study APP-induced neurodegeneration. Here we have developed an olfactory-based transgenic mouse model that overexpresses humanized-APP containing familial AD-mutations (hAPP) in either mature or immature OSNs, and found that despite the absence of extracellular plaques a striking number of apoptotic neurons were detected by 3 weeks of age. Importantly, apoptosis was restricted to the specific population overexpressing hAPP, either mature or immature OSNs, sparing those without hAPP. Interestingly, we observed that this widespread neurodegeneration could be rapidly rescued by reducing hAPP expression levels in immature neurons. Together, these data argue that overexpressing hAPP alone could induce cell-autonomous apoptosis in both mature and immature neurons, challenging the notion that amyloid plaques are necessary for neurodegeneration. Furthermore, we show that hAPP-induced neurodegeneration is reversible, suggesting that AD-related neural loss could potentially be rescued. Thus, we propose that this unique in vivo model will not only help determine the mechanisms underlying AD-related neurodegeneration but also serve as a platform to test possible treatments.
β-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer’s disease (FAD). Some FAD-based mouse models produce amyloid plaques, others don’t. β-Amyloid (Aβ) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aβ molecules aggregate in different patterns. Is there a basic cellular process governing Aβ plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of β-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aβ antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aβ antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aβ domain and the C-terminal of APP, but not co-labeled by antibodies against the Aβ C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aβ antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments.
Amyloid plaque; axonal pathology; synaptoplasticity; aging; dementia
Dominantly inherited missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, but its normal physiological function remains unclear. We previously reported that loss of LRRK2 causes impairment of protein degradation pathways as well as increases of apoptotic cell death and inflammatory responses in the kidney of aged mice.
Our analysis of LRRK2-/- kidneys at multiple ages, such as 1, 4, 7, and 20 months, revealed unique age-dependent development of a variety of molecular, cellular, and ultrastructural changes. Gross morphological abnormalities of the kidney, including altered size, weight, texture, and color, are evident in LRRK2-/- mice at 3-4 months of age, along with increased accumulation of autofluorescent granules in proximal renal tubules. The ratio of kidney/body weight in LRRK2-/- mice is increased at 1, 4, and 7 months of age (~10% at 1 month, and ~20% at 4 and 7 months), whereas the ratio is drastically decreased at 20 months of age (~50%). While kidney filtration function evaluated by levels of blood urea nitrogen and serum creatinine is not significantly affected in LRRK2-/- mice at 12-14 months of age, expression of kidney injury molecule-1, a sensitive and specific biomarker for epithelial cell injury of proximal renal tubules, is up-regulated (~10-fold). Surprisingly, loss of LRRK2 causes age-dependent bi-phasic alterations of the autophagic activity in LRRK2-/- kidneys, which is unchanged at 1 month of age, enhanced at 7 months but reduced at 20 months, as evidenced by corresponding changes in the levels of LC3-I/II, a reliable autophagy marker, and p62, an autophagy substrate. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2-/- kidneys at 7 months of age but increased at 20 months. Interestingly, the age-dependent bi-phasic alterations in autophagic activity in LRRK2-/- kidneys is accompanied by increased levels of lysosomal proteins and proteases at 1, 7, and 20 months of age as well as progressive accumulation of autolysosomes and lipofuscin granules at 4, 7-10, and 20 months of age.
LRRK2 is important for the dynamic regulation of autophagy function in vivo.
LRRK2; Parkinson's disease; knockout; LC3; p62; lysosomal proteins; cathepsins; lipofuscin
Alzheimer's disease (AD) is the most common dementia-causing disorder in the elderly, which may relate to multiple risk factors and is pathologically featured by cerebral hypometabolism, paravascular β-amyloid (Aβ) plaques, neuritic dystrophy and intra-neuronal aggregation of phosphorylated-tau. To explore potential pathogenic link among some of these lesions, we examined β-secretase-1 (BACE1) alteration relative to Aβ deposition, neuritic pathology and vascular organization in aged monkey and AD human cerebral cortex. Western blot analyses detected increased levels of BACE1 proteins and β-site-cleavage amyloid precursor protein C-terminal fragments in plaque-bearing human and monkey cortex relative to controls. In immunohistochemistry, locally elevated BACE1 immunoreactivity (IR) occurred in AD but not in control human cortex, with a trend of increased overall density among cases with greater plaque pathology. In double labeling preparations, BACE1 IR colocalized with immunolabeling for Aβ but not for phosphorylated tau. In perfusion-fixed monkey cortex, locally increased BACE1 IR co-existed with intra-axonal and extracellular Aβ IR among virtually all neuritic plaques ranging from primitive to typical cored forms. This BACE1 labeling localized to swollen/sprouting axon terminals that might co-express one or another neuronal phenotype marker (GABAergic, glutamatergic, cholinergic or catecholaminergic). Importantly, these BACE1-labeled dystrophic axons resided near or in direct contact with blood vessels. These finds implicate that plaque formation in AD or normal aging primates relate to a multisystem axonal pathogenesis that occurs in partnership with potential vascular or metabolic deficit. The data provide a tangible mechanistic explanation as to why senile plaques are present preferentially near cerebral vasculature.
Neuritic plaque; neuroplasticity; hypometabolism; aging; dementia; non-human primate
Numerous studies characterizing the function of glutathione peroxidase 4 (GPx4) have demonstrated that this selenoenzyme is protective against oxidative stress. Herein, we characterized the function of this protein by targeting GPx4 downregulation using RNA interference. Partial knockdown of GPx4 levels resulted in growth retardation and morphological changes. Surprisingly, GPx4 knockdown cells showed virtually unchanged levels of intracellular ROS, yet highly increased levels of oxidized lipid by-products. GPx1, another glutathione peroxidase and a major cellular peroxide scavenging enzyme, did not rescue GPx4-deficient cells and did not reduce lipid peroxide levels. The data established an essential role of GPx4 in protecting cells against lipid hydroperoxide damage, yet a limited role as a general antioxidant enzyme. As oxidized lipid hydroperoxides are a characteristic of neurodegenerative diseases, we analyzed brain tissues of mice suffering from a model of Alzheimer's disease and found that oxidized lipid by-products were enriched, and expression of both GPx4 and guanine-rich sequence-binding factor, which is known to control GPx4 synthesis, was downregulated. Brain tissue from an Alzheimer's diseased human also manifested enhanced levels of one of the oxidized lipid by-products, 4-hydroxynonenal. These data suggest a role of GPx4 in neurodegenerative diseases through its function in removal of lipid hydroperoxides. Antioxid. Redox Signal. 12, 819–827.