Glucocerebrosidase (GCase) functions as a lysosomal enzyme and its mutations are known to be related to many neurodegenerative diseases, including Gaucher's disease (GD), Parkinson's disease (PD), and Dementia with Lewy Bodies (DLB). However, there is little information about the role of GCase in the pathogenesis of Alzheimer's disease (AD). Here we demonstrate that GCase protein levels and enzyme activity are significantly decreased in sporadic AD. Moreover, Aβ1-42 oligomer treatment results in neuronal cell death that is concomitant with decreased GCase protein levels and enzyme activity, as well as impairment in lysosomal biogenesis and acidification. Importantly, overexpression of GCase promotes the lysosomal degradation of Aβ1-42 oligomers, restores the lysosomal impairment, and protects against the toxicity in neurons treated with Aβ1-42 oligomers. Our findings indicate that a deficiency of GCase could be involved in progression of AD pathology and suggest that augmentation of GCase activity may be a potential therapeutic option for the treatment of AD.

Heterozygous mutations in glucocerebrosidase 1 (GBA1) are a major genetic risk factor for Parkinson's disease and Dementia with Lewy bodies. Mutations in GBA1 leads to GBA1 enzyme deficiency, and GBA1-associated parkinsonism has an earlier age of onset and more progressive parkinsonism. To investigate a potential influence of GBA1 deficiency caused by mutations in GBA1 on the disease progression of PD, GBA1 mice carrying D409H knock-in mutation were crossbred with the human A53T (hA53T) α-synuclein transgenic mice. Here, we show that GBA1 enzyme activity plays a significant role in the hA53T α-synuclein induced α-synucleinopathy. The expression of D409H GBA1 markedly shortens the lifespan of hA53T α-synuclein transgenic mice. Moreover, D409H GBA1 expression exacerbates the formation of insoluble aggregates of α-synuclein, glial activation, neuronal degeneration, and motor abnormalities in the hA53T α-synuclein transgenic mice. Interestingly, the expression of D409H GBA1 results in the loss of dopaminergic neurons in the substantia nigra pars compacta of hA53T transgenic mice. Taken together, these results indicate that GBA1 deficiency due to D409H mutation affects the disease onset and course in hA53T α-synuclein transgenic mice. Therefore, strategies aimed to maintain GBA1 enzyme activity could be employed to develop an effective novel therapy for GBA1 linked-PD and related α-synucleinopathies.

Alpha-synuclein (α-SYN) is one of the key contributors in Parkinson's disease (PD) pathogenesis. Despite the fact that increased α-SYN levels are considered one of the key contributors in developing PD, the molecular mechanisms underlying the regulation of α-SYN still needs to be elucidated. Since the 3' untranslated regions (3'UTRs) of messenger RNAs (mRNAs) have important roles in translation, localization, and stability of mRNAs through RNA binding proteins (RBPs) and microRNAs (miRNAs), it is important to identify the exact length of 3'UTRs of transcripts in order to understand the precise regulation of gene expression. Currently annotated human α-SYN mRNA has a relatively long 3'UTR (2529 nucleotides [nt]) with several isoforms. RNA-sequencing and epigenomics data have suggested, however, the possible existence of even longer transcripts which extend beyond the annotated α-SYN 3'UTR sequence. Here, we have discovered the novel extended form of α-SYN 3'UTR (3775 nt) in the substantia nigra of human postmortem brain samples, induced pluripotent stem cell (iPSC)-derived dopaminergic neurons, and other human neuronal cell lines. Interestingly, the longer variant reduced α-SYN translation. The extended α-SYN 3'UTR was significantly lower in iPSC-derived dopaminergic neurons from sporadic PD patients than controls. On the other hand, α-SYN protein levels were much higher in PD cases, showing the strong negative correlation with the extended 3'UTR. These suggest that dysregulation of the extended α-SYN 3'UTR might contribute to the pathogenesis of PD.

Heterozygous mutations in GBA, the gene encoding the lysosomal enzyme glucosylceramidase beta/β-glucocerebrosidase, comprise the most common genetic risk factor for Parkinson disease (PD), but the mechanisms underlying this association remain unclear. Here, we show that in GbaL444P/WT knockin mice, the L444P heterozygous Gba mutation triggers mitochondrial dysfunction by inhibiting autophagy and mitochondrial priming, two steps critical for the selective removal of dysfunctional mitochondria by autophagy, a process known as mitophagy. In SHSY-5Y neuroblastoma cells, the overexpression of L444P GBA impeded mitochondrial priming and autophagy induction when endogenous lysosomal GBA activity remained intact. By contrast, genetic depletion of GBA inhibited lysosomal clearance of autophagic cargo. The link between heterozygous GBA mutations and impaired mitophagy was corroborated in postmortem brain tissue from PD patients carrying heterozygous GBA mutations, where we found increased mitochondrial content, mitochondria oxidative stress and impaired autophagy. Our findings thus suggest a mechanistic basis for mitochondrial dysfunction associated with GBA heterozygous mutations. Abbreviations: AMBRA1: autophagy/beclin 1 regulator 1; BECN1: beclin 1, autophagy related; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chloroyphenylhydrazone; CYCS: cytochrome c, somatic; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; GBA: glucosylceramidase beta; GBA-PD: Parkinson disease with heterozygous GBA mutations; GD: Gaucher disease; GFP: green fluorescent protein; LC3B: microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated form of microtubule-associated protein 1 light chain 3 beta; MitoGreen: MitoTracker Green; MitoRed: MitoTracker Red; MMP: mitochondrial membrane potential; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH transcription factor; NBR1: NBR1, autophagy cargo receptor; Non-GBA-PD: Parkinson disease without GBA mutations; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; ROS: reactive oxygen species; SNCA: synuclein alpha; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1/Porin: voltage dependent anion channel 1; WT: wild type.

Mutations in glucocerebrosidase (GBA) cause Gaucher disease (GD) and increase the risk of developing Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Since both genetic and environmental factors contribute to the pathogenesis of sporadic PD, we investigated the susceptibility of nigrostriatal dopamine (DA) neurons in L444P GBA heterozygous knock-in (GBA +/L444P ) mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective dopaminergic mitochondrial neurotoxin.

Analysis of human pathology led Braak to postulate that α-synuclein (α-syn) pathology could spread from the gut to brain via the vagus nerve. Here, we test this postulate by assessing α-synucleinopathy in the brain in a novel gut-to-brain α-syn transmission mouse model, where pathological α-syn preformed fibrils were injected into the duodenal and pyloric muscularis layer. Spread of pathologic α-syn in brain, as assessed by phosphorylation of serine 129 of α-syn, was observed first in the dorsal motor nucleus, then in caudal portions of the hindbrain, including the locus coeruleus, and much later in basolateral amygdala, dorsal raphe nucleus, and the substantia nigra pars compacta. Moreover, loss of dopaminergic neurons and motor and non-motor symptoms were observed in a similar temporal manner. Truncal vagotomy and α-syn deficiency prevented the gut-to-brain spread of α-synucleinopathy and associated neurodegeneration and behavioral deficits. This study supports the Braak hypothesis in the etiology of idiopathic Parkinson's disease (PD).

The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes β-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.

Mutations and loss of activity in PARKIN, an E3 ubiquitin ligase, play a role in the pathogenesis of Parkinson's disease (PD). PARKIN regulates many aspects of mitochondrial quality control including mitochondrial autophagy (mitophagy) and mitochondrial biogenesis. Defects in mitophagy have been hypothesized to play a predominant role in the loss of dopamine (DA) neurons in PD. Here, we show that although there are defects in mitophagy in human DA neurons lacking PARKIN, the mitochondrial deficits are primarily due to defects in mitochondrial biogenesis that are driven by the upregulation of PARIS and the subsequent downregulation of PGC-1α. CRISPR/Cas9 knockdown of PARIS completely restores the mitochondrial biogenesis defects and mitochondrial function without affecting the deficits in mitophagy. These results highlight the importance mitochondrial biogenesis versus mitophagy in the pathogenesis of PD due to inactivation or loss of PARKIN in human DA neurons.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an unambiguous cause of late-onset, autosomal-dominant familial Parkinson's disease (PD) and LRRK2 mutations are the strongest genetic risk factor for sporadic PD known to date. A number of transgenic mice expressing wild-type or mutant LRRK2 have been described with varying degrees of LRRK2-related abnormalities and modest pathologies. None of these studies directly addressed the role of the kinase domain in the changes observed and none of the mice present with robust features of the human disease. In an attempt to address these issues, we created a conditional LRRK2 G2019S (LRRK2 GS) mutant and a functionally negative control, LRRK2 G2019S/D1994A (LRRK2 GS/DA). Expression of LRRK2 GS or LRRK2 GS/DA was conditionally controlled using the tet-off system in which the presence of tetracycline-transactivator protein (tTA) with a CAMKIIα promoter (CAMKIIα-tTA) induced expression of TetP-LRRK2 GS or TetP-LRRK2 GS/DA in the mouse forebrain. Overexpression of LRRK2 GS in mouse forebrain induced behavioral deficits and α-synuclein pathology in a kinase-dependent manner. Similar to other genetically engineered LRRK2 GS mice, there was no significant loss of dopaminergic neurons. These mice provide an important new tool to study neurobiological changes associated with the increased kinase activity from the LRRK2 G2019S mutation, which may ultimately lead to a better understanding of not only the physiologic actions of LRRK2, but also potential pathologic actions that underlie LRRK2 GS-associated PD.

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.

Activation of microglia by classical inflammatory mediators can convert astrocytes into a neurotoxic A1 phenotype in a variety of neurological diseases1,2. Development of agents that could inhibit the formation of A1 reactive astrocytes could be used to treat these diseases for which there are no disease-modifying therapies. Glucagon-like peptide-1 receptor (GLP1R) agonists have been indicated as potential neuroprotective agents for neurologic disorders such as Alzheimer's disease and Parkinson's disease3-13. The mechanisms by which GLP1R agonists are neuroprotective are not known. Here we show that a potent, brain-penetrant long-acting GLP1R agonist, NLY01, protects against the loss of dopaminergic neurons and behavioral deficits in the α-synuclein preformed fibril (α-syn PFF) mouse model of sporadic Parkinson's disease14,15. NLY01 also prolongs the life and reduces the behavioral deficits and neuropathological abnormalities in the human A53T α-synuclein (hA53T) transgenic mouse model of α-synucleinopathy-induced neurodegeneration16. We found that NLY01 is a potent GLP1R agonist with favorable properties that is neuroprotective through the direct prevention of microglial-mediated conversion of astrocytes to an A1 neurotoxic phenotype. In light of its favorable properties, NLY01 should be evaluated in the treatment of Parkinson's disease and related neurologic disorders characterized by microglial activation.

Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl-deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.

Alzheimer's disease (AD) is the most common cause of age-related dementia. Increasing evidence suggests that neuroinflammation mediated by microglia and astrocytes contributes to disease progression and severity in AD and other neurodegenerative disorders. During AD progression, resident microglia undergo proinflammatory activation, resulting in an increased capacity to convert resting astrocytes to reactive astrocytes. Therefore, microglia are a major therapeutic target for AD and blocking microglia-astrocyte activation could limit neurodegeneration in AD. Here we report that NLY01, an engineered exedin-4, glucagon-like peptide-1 receptor (GLP-1R) agonist, selectively blocks β-amyloid (Aβ)-induced activation of microglia through GLP-1R activation and inhibits the formation of reactive astrocytes as well as preserves neurons in AD models. In two transgenic AD mouse models (5xFAD and 3xTg-AD), repeated subcutaneous administration of NLY01 blocked microglia-mediated reactive astrocyte conversion and preserved neuronal viability, resulting in improved spatial learning and memory. Our study indicates that the GLP-1 pathway plays a critical role in microglia-reactive astrocyte associated neuroinflammation in AD and the effects of NLY01 are primarily mediated through a direct action on Aβ-induced GLP-1R+ microglia, contributing to the inhibition of astrocyte reactivity. These results show that targeting upregulated GLP-1R in microglia is a viable therapy for AD and other neurodegenerative disorders.

Emerging evidence indicates that cellular senescence could be a critical inducing factor for aging-associated neurodegenerative disorders. However, the involvement of cellular senescence remains unclear in Parkinson's disease (PD). To determine this, we assessed the effects of α-synuclein preformed fibrils (α-syn PFF) or 1-methyl-4-phenylpyridinium (MPP+) on changes in cellular senescence markers, employing α-syn PFF treated-dopaminergic N27 cells, primary cortical neurons, astrocytes and microglia and α-syn PFF-injected mouse brain tissues, as well as human PD patient brains. Our results demonstrate that α-syn PFF-induced toxicity reduces the levels of Lamin B1 and HMGB1, both established markers of cellular senescence, in correlation with an increase in the levels of p21, a cell cycle-arrester and senescence marker, in both reactive astrocytes and microglia in mouse brains. Using Western blot and immunohistochemistry, we found these cellular senescence markers in reactive astrocytes as indicated by enlarged cell bodies within GFAP-positive cells and Iba1-positive activated microglia in α-syn PFF injected mouse brains. These results indicate that PFF-induced pathology could lead to astrocyte and/or microglia senescence in PD brains, which may contribute to neuropathology in this model. Targeting senescent cells using senolytics could therefore constitute a viable therapeutic option for the treatment of PD.

Mutations in the GBA1 gene have been identified as a prevalent genetic risk factor for Parkinson's disease (PD). GBA1 mutations impair enzymatic activity, leading to lysosomal dysfunction and elevated levels of α-synuclein (α-syn). While most research has primarily focused on GBA1's role in promoting synucleinopathy, emerging evidence suggests that neuroinflammation may be a key pathogenic alteration caused by GBA1 deficiency. To examine the molecular mechanism underlying GBA1 deficiency-mediated neuroinflammation, we generated Gba1 E326K knock-in (KI) mice using the CRISPR/Cas9 technology, which is linked to an increased risk of PD and dementia with Lewy bodies (DLB). In the ventral midbrain and hippocampus of 24-month-old Gba1 E326K KI mice, we found a moderate decline in GBA1 enzymatic activity, a buildup of glucosylceramide, and an increase in microglia density. Furthermore, we observed increased levels of pro-inflammatory cytokines and formation of reactive astrocytes in primary microglia and astrocytes, respectively, cultured from Gba1 E326K KI mice following treatment with pathologic α-syn preformed fibrils (PFF). Additionally, the gut inoculation of α-syn PFF in Gba1 E326K KI mice significantly enhanced the accumulation of Lewy bodies in the dentate gyrus of the hippocampus, accompanied by aggravated neuroinflammation and exacerbated non-motor symptoms. This research significantly enhances our understanding of the Gba1 E326K mutation's involvement in neuroinflammation and the cell-to-cell transmission of pathogenic α-syn in the brain, thereby opening new therapeutic avenues.

Accumulating evidence suggests that the non-receptor tyrosine kinase c-Abl plays an important role in the progression of Parkinson's disease (PD) and c-Abl inhibition could be neuroprotective in PD and related α-synucleinopathies. Nilotinib, a c-Abl inhibitor, has shown improved motor and cognitive symptoms in PD patients. However, issues concerning blood-brain barrier (BBB) penetration, lack of selectivity and safety still remain. Radotinib HCl is a selective Bcr-Abl kinase inhibitor that not only effectively access the brain, but also exhibits greater pharmacokinetic properties and safety profiles compared to Nilotinib and other c-Abl inhibitors. Here, we show the neuroprotective efficacy of Radotinib HCl, a brain penetrant c-Abl inhibitor, in a pre-clinical model of PD. Importantly, in vitro studies demonstrate that the treatment of Radotinib HCl protects the α-synuclein preformed fibrils (PFF)-induced neuronal toxicity, reduces the α-synuclein PFF-induced Lewy bodies (LB)/Lewy neurites (LN)-like pathology and inhibits the α-synuclein PFF-induced c-Abl activation in primary cortical neurons. Furthermore, administration of Radotinib HCl inhibits c-Abl activation and prevents dopaminergic neuron loss, neuroinflammation and behavioral deficits following α-synuclein PFF-induced toxicity in vivo. Taken together, our findings indicate that Radotinib HCl has beneficial neuroprotective effects in PD and provides an evidence that selective and brain permeable c-Abl inhibitors can be potential therapeutic agents for the treatment of PD and related α-synucleinopathies.

Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna's E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna's PAR binding and Iduna's E3 ligase activity. Iduna's E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna's PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair.

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson's disease are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic Parkinson's disease. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide, termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and are investigated by site-directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with Parkinson's disease, suggesting that this loss may be pathologic. This implies that hydrogen sulfide donors may be therapeutic.

Vacuolar protein sorting-associated protein 35 (VPS35) is involved in retrograde transport of proteins from endosomes to trans-Golgi network. Gene mutations in VPS35 are linked to autosomal dominant late-onset Parkinson's disease (PD). Although the identification of VPS35 mutations has provided novel insight about its interactions with several PD-associated genes including leucine-rich repeat kinase 2 (LRRK2) and α-synuclein, little information is available about the molecular mechanisms of cell death downstream of VPS35 dysfunction. In this study, we showed that VPS35 has a role in the lysosomal degradation of parkin substrate aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2), of which accumulation leads to poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death. VPS35 was co-immunoprecipitated with AIMP2, as well as lysosome-associated membrane protein-2a (Lamp2a). Interestingly, this association was disrupted by PD-associated VPS35 mutant D620N. VPS35 overexpression prevented AIMP2-potentiated cell death and PARP1 activation in SH-SY5Y cells. More importantly, knockdown of VPS35 led to PARP1 activation and cell death, which was AIMP2 dependent. These findings provide new mechanistic insights into the role of VPS35 in the regulation of AIMP2 levels and cell death. As AIMP2 accumulation was reported in PD patient's brains and involved in dopaminergic cell death, identification of VPS35 as a novel regulator of AIMP2 clearance via lysosomal pathway provides alternative venue to control dopaminergic cell death in PD.

c-Abl is activated in the brain of Parkinson's disease (PD) patients and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice where it inhibits parkin through tyrosine phosphorylation leading to the accumulation of parkin substrates, and neuronal cell death. In the present study, we evaluated the in vivo efficacy of nilotinib, a brain penetrant c-Abl inhibitor, in the acute MPTP-induced model of PD. Our results show that administration of nilotinib reduces c-Abl activation and the levels of the parkin substrate, PARIS, resulting in prevention of dopamine (DA) neuron loss and behavioral deficits following MPTP intoxication. On the other hand, we observe no reduction in the tyrosine phosphorylation of parkin and the parkin substrate, AIMP2 suggesting that the protective effect of nilotinib may, in part, be parkin-independent or to the pharmacodynamics properties of nilotinib. This study provides a strong rationale for testing other brain permeable c-Abl inhibitors as potential therapeutic agents for the treatment of PD.