Niemann-Pick Type C Disease: At the Nexus of Neurodegenerative and Neurodevelopmental Disorders

Rare genetic diseases can provide valuable insights into more common disorders by linking specific genes and pathways to shared disease phenotypes. The rare Niemann-Pick Type C disease (NPC) is a neurological disorder that has often been compared to Alzheimer’s Disease (AD) because both diseases are characterized by cognitive impairment in the presence of tau pathology and altered Amyloid Precursor Protein (APP) processing and Aβ metabolism. Here we review the molecular pathology of NPC and critically examine the similarities between NPC, AD and other neurological disorders. Besides the phenotypic overlap between AD and NPC, there is substantial evidence that cholesterol metabolism is altered in both diseases. Specifically, the epsilon 4 allele of the brain cholesterol transport protein Apolipoprotein E (ApoE4 ) is the strongest risk factor for late onset AD (LOAD) whereas NPC disease is caused by point mutations in the cholesterol transport proteins NPC1 and NPC2. In contrast to AD, NPC encompasses a broad neurovisceral disease phenotype having a diversity of penetrance, age of onset, and both central and peripheral manifestations. In addition to features that are in common with AD, NPC frequently exhibits close phenotypic overlap with neurodevelopmental disorders such as schizophrenia. Understanding the mechanistic links shared by NPC, AD, and neurodevelopmental disorders should enable a more holistic approach to therapeutic strategies to diseases which superficially appear very different.


Introduction
Neurodevelopmental and neurodegenerative diseases are generally thought to differ fundamentally in cause, course of disease and disease phenotype. Neurodevelopmental diseases arise from perturbed development of the nervous system while neurodegenerative diseases emerge from chronic degenerative changes in the brain resulting from stress, injury, altered metabolism or other maladaptive processes. As will be discussed in detail below, the Niemann-Pick disease type C (NPC) clinical phenotype includes psychosis and dementia [1,2], features of the common neurodevelopmental disease schizophrenia and the most common neurodegenerative disease Alzheimer's Disease (AD) [3,4], respectively. NPC results from mutations in either of two cholesterol transport proteins, NPC1, or NPC2, highlighting the fundamental role played by cholesterol homeostasis in brain function and disease [5,6]. These neurodevelopmental and neurodegenerative processes may engage components of lipid metabolism in reciprocal ways frustrating the therapeutic targeting of many pathway components however, comparing the NPC phenotype to AD brings to light symptoms not normally associated with AD, such as psychosis [7][8][9]. This suggests that certain AD features or population subtypes may have characteristics of neurodevelopmental diseases such as alterations in developmental signaling pathways [10][11][12][13][14].
Page -02 ISSN: 2376-922X NPC mutations [25]. Genetic screening studies reveal that a late onset phenotype might be present with a much higher incidence, between 1:19,000-1:36,000 [26], and may be overrepresented and under diagnosed among adults with neurological and psychiatric symptoms [27]. These findings suggest that there is a wealth of opportunity to explore modulators of disease severity.

Molecular Pathology of NPC (Figure 1)
Both NPC1 and NPC2 work in concert as cholesterol binding proteins that regulate transport of LDL-derived cholesterol to the endosome and from the late endosome to various intracellular targets including the endoplasmic reticulum, lysosome, Golgi and mitochondria [28,29]. Expression of both proteins is regulated by cholesterol levels via SREBP pathways.
NPC1 is an intrinsic membrane protein having an N-Terminal Domain (NTD), three luminal domains and 13 transmembrane helices several of which comprise a sterol sensing domain [30]. NPC1 mutations alter not only cholesterol binding and transport functions but also expression levels, processing and localization in a mutationrelated manner [31,32]. NPC1 protein has significant homology to the Patched1 (Ptc1) morphogen receptor that is part of the Hedgehog (Shh) pathway [33] and, along with Ptc1, significant homology to the resistance-nodulation-division (RND) family of permeases suggesting a role in fatty acid and multidrug transport [34].
The NPC2 protein is a soluble protein that was originally identified

NPC Protein Interactions and Cellular Pathways (Figure 2)
The expression and protein stability of NPC proteins are influenced by numerous interacting proteins suggesting possible strategies for therapeutic intervention through the manipulation of NPC protein levels.

NPC1
Levels of NPC1 are regulated by TMEM97/σ2 receptor [46]. Knockdown of TMEM97/σ2 increases NPC1 protein levels in cell culture but anti-sense oligomers (ASOs) to TMEM97/σ2 failed to influence NPC1 levels in vivo in rat liver. It is unclear whether brain NPC1 levels would have responded if brain penetrant ASOs had been employed. It has been suggested that TMEM97/σ2 may act as a chaperone protein for NPC1 limiting its generation and export from the endoplasmic reticulum (ER) TMEM97/σ2 is itself a robust target for neuropsychiatric compounds including haloperidol, ketamine, methamphetamine and phencyclidine (all agonists).
TMEM97/σ2 is involved in one of several complexes with the low-density lipoprotein receptor (LDLR) upstream of NPC1 that may be differentially regulated among different tissues. TMEM97/σ2 forms a complex with progesterone receptor membrane component 1 (PGRMC1) and LDLR to promote internalization of LDL [47]. LDLR also forms clathrin-dependent internalization complexes with proprotein convertase subtilisin/kexin-9 (PCSK-9) and the adaptor protein autosomal hypercholesterolemia (ARH)/ receptor associated protein (LDLRAP). LDLRAP protein levels are high in liver but low in brain and may provide an alternative to TMEM97/σ2 for LDLR internalization in liver. Transcriptional profiling of NPC1 knockout mice links NPC1 to levels of tau, apolipoprotein C1 (ApoC1), sortilin 1, nexins 12, 13, 17, and ATP-binding cassette sub-family A (ABCA) members 2, 5, and 8B which are all related to active targets for intervention in AD. ABCA2, for example, is reported to regulate amyloid precursor protein (APP) expression via sphingolipid metabolism [54,55]. APP protein increases in cerebellum and hippocampus of NPC1 knockout mice [56]. Reduction of NPC1 levels by proteasomal degradation [57], is a consequence of Akt activation. NPC1 levels appear to be reciprocally regulated by TMEM97/σ2 receptor. TMEM97 forms a complex with PGRMC1 and LDLR. LDLR levels are enhanced by NDRG1, and down regulated by IDOL (left). NPC1 directly interacts with SLC38A9 to mediate cholesterol activation of mTORC1 which in turn regulates SREBP activity. Activation of SREBP by SCAP yields an amino-terminal fragment which translocates to the nucleus to activate transcription of NPC1 and NPC2 genes (right). NPC2 levels are stabilized by Nogo B receptor (left) while NPC1L1 protein promotes its degradation. NPC1 deficiency promotes NPC2 levels and vice versa.

Relationship to Cholesterol Sensing by mTORC1
NPC1 forms a complex with the lysosomal transmembrane protein SLC38A9 which mediates cholesterol activation of mTOR complex 1 (mTORC1) [61]. mTORC1 regulates sterol regulatory element binding proteins (SREBP)1 and SREBP2 activity [62,63]. mTOR is a link between AD, in which mTOR is chronically activated with detrimental impact on autophagy and tau phosphorylation [64][65][66], and schizophrenia which is characterized by hypofunction of the mTOR pathway [67].

NPC Proteins as pharmacological targets
NPC protein levels are sensitive to treatment with amphiphilic psychotropic and antidepressant drugs [68,69]. The cationic amphiphile U18666A binds to NPC1 [70], inhibits cholesterol binding and recapitulates features of NPC disease phenotype [71]. The antidepressant amitriptyline induces the accumulation of cytoplasmic cholesterol levels and increases expression of NPC2 mRNA [69].
Amitriptyline treatment increases the secretion of NPC2, causes neurogenesis and improves cognition in 3XTg Alzheimer's mice [72]. It also causes functional improvement in a Huntington's disease mouse model via increased neurotrophin signaling [73]. Amitriptyline has also shown benefit in the context of another neurodegenerative disease, progressive supranuclear palsy (PSP) [74,75].

Current treatment strategies
Efforts to standardize disease diagnosis and treatment strategies have been reported [76]. Diagnosis of NPC disease typically involves histopathological analysis using filipin, a fluorescent macrolide antibiotic that binds to cholesterol. The compound has also been used extensively for chemical screening of compounds for the treatment of NPC [77][78][79][80]. A positive filipin test would prompt genetic testing for NPC1 or NPC2 mutations. Specific blood oxysterol profiles are associated with NPC disease [81]. The Phase 1-2 clinical trial for Intracerebroventricular (ICV) β-cyclodextrin employed plasma hydroxycholesterol [82], cerebrospinal fluid (CSF) fatty acid binding protein (FABP) and calbindin, a marker for Purkinje cell degeneration as biomarkers. In addition, various magnetic resonance modalities such as MRI have been employed as imaging biomarkers [83,84].
Robust treatment approaches include ICV injections of β-cyclodextrin (BCD) which acts essentially as a cholesterol chaperone. BCD treatment is the result of novel research collaboration between academic, government and industry researchers and family members called Support of Accelerated Research-NPC (SOAR-NPC) [85].
NPC is associated not only with accumulation of cholesterol, but also with sphingosine, sphingomyelin and glycosphingolipids (GSL's) resulting in altered endolysosomal calcium homeostasis as a result of inhibiting the mucolipin TRP channel 1 (TRPML1) [90][91][92][93]. The glycosylceramide synthase inhibitor Miglustat is approved for the treatment of NPC and reportedly stabilizes or improves neurological manifestations [76,[94][95][96]. In the feline NPC model, the adverse neurological phenotype was delayed with miglustat treatment without having a significant impact on cholesterol accumulation or visceral endpoints suggesting that neurological manifestations and cholesterol accumulation, as well as central and peripheral manifestations are separable phenomena [97].
Treatment with fingolimod (FTY720), a sphingosine analog and sphingosine-1-phosphate receptor agonist, increases NPC1 and NPC2 expression, and reduces both cholesterol and sphingolipids in NPC mutant cells [98]. Fingolimod, becomes a potent histone deacetylase (HDAC) inhibitor once phosphorylated. It is being evaluated in clinical trials for NPC disease and has been tested in the context of AD models [99]. Dysregulation of sphingolipid metabolism is observed in AD where it correlates with CSF Aβ levels and contributes to impairment of autophagy [100][101][102][103].
Cellular treatment with sphingolipids causes a "molecular trap" for cholesterol Sphingolipid treatment results in SREBP cleavage by SREBP cleavage-activating protein (SCAP) [48], and subsequent upregulation of LDL receptors which is the source of the elevated cholesterol. LDL receptors as well as TMEM97/σ2, a protein that interacts with, and might regulate NPC1 levels are targets of SREBP [104]. In turn the lipogenic activity of SREBP1 is regulated by mTORC1 and promotes cell growth via Akt signaling [62].

NPC and AD (Table 2)
The observation of AD-like neurofibrillary tangles and diffuse amyloid deposits in NPC have prompted numerous studies to search for molecular links between these two diseases. For example, while there is little evidence for a direct genetic relationship between NPC and AD, there is reported epistasis between NPC1 and ATPbinding cassette type A1 (ABCA1) and AD risk [105]. ABCA1 is a critical lipidating gene for ApoE. ApoE4 is the strongest risk factor for sporadic AD and has been found to be poorly lipidated compared to the other common human ApoE isoforms, ApoE2 and ApoE3 [106]. Increasing ApoE4 lipidation has been suggested as a therapeutic strategy for AD. It has been reported that NPC disease patients have dysregulated ABCA1 expression and reduced ABCA1 activity [107].
The NPC1 inhibitor U18666A, not only inhibits NPC1 cholesterol binding and recapitulate features of NPC disease phenotype [71], treatment with the drug alters APP metabolism resulting in endosomal -lysosomal processing of APP [108]. Knockout of the NPC1 gene has similar effects in vivo all suggesting that NPC proteins can influence the amyloidolytic processing of APP. Levels of NPC1 are increased in Alzheimer's disease and in APP/ PS1 transgenic animals [56,109], a surprising finding if the AD phenotype in NPC disease is thought to be the result of NPC1 or NPC2 loss of function. It is possible that such elevations in NPC protein levels reflect a homeostatic response Several additional studies have employed animal models of NPC disease (reviewed by [110]) crossed with models of AD. Deletion of either Tau or APP exacerbates the NPC phenotype [111,112].
The deletion of tau is thought to impair the cytoplasmic transport required for autophagy, while the exacerbation of phenotype caused by the loss of APP suggests that APP may play a compensatory role for the loss of cholesterol transport proteins. Aβ is reported to have a role in regulating lipid homeostasis and furthermore lipid-associated Aβ is increased in NPC suggesting a potential lipid "chaperone" role for the peptide. In contrast to APP deletion [113,114], APP overexpression in NPC-deficient background yields increased Aβ generation and the production of shortened γ-C-terminal fragments (γ-CTFs) suggesting correspondingly longer and potentially more toxic forms of Aβ [114][115][116].
APP, APP fragments and APP processing enzymes interact robustly with SREBP2, which in turn regulates NPC expression. Aβ and β-cleaved APP inhibit SREBP2, while α-soluble APP stimulates it [117,118]. The nuclear translocation of SREBP2 N-terminal fragments, which is required for SREBP transcriptional activation is impaired in AD and tau transgenic animals, but not in APP transgenic animals suggesting that AD-related tau dys-homeostasis can alter SREBP2 signaling [119]. Similarly, dysregulation of SREBP2 caused by high cholesterol conditions can cause an increase in the expression of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) under the same conditions expected to result in an increase in NPC expression [120].

NPC and other neurodegenerative diseases
NPC is a lysosomal storage disease, a category of neurodegenerative disorders that includes the sphingolipidoses. This class of disorders is characterized by aberrations in sphingolipid metabolism and includes Niemann-Pick disease types A and B caused by defects in sphingomyelinase (SMPD1), Gaucher's disease caused by galactosidase (GBA1) deficiency, Fabry's disease associated with α-galactosidase-A (GLA) deficiency, Krabbe disease associated with galactosidase (GALC) deficiency, Tay-Sachs caused by β-hexosaminidase-A mutations and metachromatic leukodystrophy (MLD) resulting from defects in arylsulfatase A (ARSA) [121]. Although cholesterol is the principle material impacted by NPC mutations, they also influence sphingolipid metabolism and therefore are included in this class of disorders all of which have devastating neurological consequences.
Due to the essential role NPC proteins play in cholesterol metabolism, it is not surprising that impairment of their function results in phenotypic overlap with numerous neurological diseases such as fronto-temporal dementia (FTD), Parkinson's disease, multiple sclerosis (MS) and other inflammatory disorders. The presence of frontal lobe atrophy suggests similarities with FTD [36]. A potential link with FTD is further supported by the observations that there is aberrant cytoplasmic localization of the FTD-related protein TAR DNA-binding protein 43 (TDP-43) in NPC models, and that the expression of TDP-43 regulated genes such as transcription factor AP-2 alpha (TFAP2A), ciliary neurotrophic factor receptor (CNTFR), MAP kinase-activating death domain protein (MADD), myocyte-specific enhancer factor 2D (MEF2D), transducin-like enhancer protein 1 (TLE1) and TRAF2 and NCK-interacting protein kinase (TNIK) is altered [122].
Parkinsonism is associated with NPC heterozygosity [25], and NPC cases share features of synucleinopathy associated with PD including Lewy bodies and Lewy neurites as detected by immunoreactivity for phosphorylated synuclein [20]. Ceramide metabolism appears to be perturbed in both NPC and PD [90,91,98]. Mutations in GBA1 cause Gaucher's disease when homozygous, or a predisposition to PD when heterozygous [123,124]. The accumulation of GSLs caused by GBA1 mutations can be mitigated with the pharmacological chaperone afegostat-tartrate (isofagomine) or with inhibitors of glycosylceramide synthase such as inhibitor GZ667161, which has been tested in models of synucleinopathy [125,126], and miglustat, which has been employed in NPC models and human NPC subjects as discussed above. Impaired mitophagy has been implicated in PD pathogenesis. NPC2 as well as the PD related genes parkin and PTEN-Induced kinase (PINK1) are regulators of mitochondrial autophagy suggesting a mechanistic link between NPC and PD [40,127].
Cholesterol is a major component of myelin, so a relationship between diseases that result from dysregulation of cholesterol homeostasis and demyelinating diseases is expected. A case of adult NPC disease originally diagnosed as multiple sclerosis and a report of severe demyelination in a case of juvenile NPC disease illustrate the connection between aberrant cholesterol metabolism and impaired myelination of neurons. Defects in myelination are common both in human NPC disease and in the knockout mouse model and proteomic studies of the corpus callosum from knockout mice have identified specific factors involved in defective myelination including glycolipid transfer protein (GLTP), ceramide synthase 2 (CerS2), and 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (UGT8). Fingolamod, a therapeutic approved for the treatment of MS is under evaluation for its utility in NPC disease. In the context of inflammation, NPC1 mutations are associated with activation of the innate immune system and chronic inflammation [128][129][130][131][132], however NPC2 knockdown reduced lipopolysaccharide (LPS)induced expression of pro-inflammatory genes suggesting Toll-like receptors (TLR) signaling activation requires NPC2 [40].

NPC and other neurodevelopmental disorders
In contrast to the features that are conventionally associated with AD, NPC features psychosis as a component of disease phenotype, and stereotypy is a feature of both psychosis and NPC. Both NPC disease and schizophrenia are associated with cerebellar impairment [133][134][135]. Likewise seizure is a feature of NPC disease and AD. In a study on glutamatergic function in NPC1 -/-mice, AMPA receptors did not respond to prolonged application of agonist to cause a reduction in synaptic transmission despite normal AMPA receptor protein levels [136]. Similarly, studies on iPSC-derived NPC1 mutant neurons show upregulation of AMPA receptor expression and protein level, but attenuated function. Collectively, these data suggest that NPC protein plays an important role glutamatergic function.
As mentioned, TMEM97/σ2 is both a molecular partner of NPC1 and a robust target for antipsychotic medications suggesting a link Page -06 ISSN: 2376-922X between NPC and psychiatric disorders, while other psychotropic drugs upregulate expression of NPC1, NPC2 and other cholesterol transport genes through regulation of SREBP [68]. Large numbers of undiagnosed NPC mutations among psychiatric patients have been reported suggesting that psychosis may be a major manifestation of adult onset NPC disease [27,137,138].

The neurodevelopmental -neurodegenerative disorder overlap: Alzheimer's disease + psychosis
If cholesterol metabolism is truly central to pathogenesis in both NPC and AD, it suggests that there may be additional phenotypic overlaps which are less commonly observed. Psychosis, for example, is a feature of NPC, and distinguishes an AD subtype. AD plus Psychosis (AD+P) is now recognized be associated with accelerated cognitive decline, hypofrontality and a significant (as much as 61%) heritability [7,8,139,140]. Psychosis is reported in as many as 50% of individuals with AD and is associated with greater cortical synaptic impairment [9]. In the Tg4510 mouse model (P301L mutant human Tau), a psychosis phenotype (pre-pulse inhibition, PPI) correlates with brain load of hyperphosphorylated tau [141].
Treatment with the anti-psychotic haloperidol reduces tau phosphorylation in the same model by inhibiting AMPK consistent with postmortem human observation of reduced neurofibrillary load in subjects treated with haloperidol [142][143][144]. A psychotic phenotype was also described in the APPswe/PSI deltaE9 transgenic model, and rescued by knockdown of a protein linked to schizophrenia, kalirin [145].
ApoE genotype appears to correlate with both the occurrence of psychosis in AD and with the presence of Lewy body pathology, with those carrying two ApoE4 alleles at greatest risk [146,147].

Discussion and Conclusion
The NPC proteins regulate the critical transit of cholesterol through the endocytic pathway and as such appear to be prime targets for interventions into many pathogenic processes whether developmental or degenerative in origin [148]. The expression and stability of the NPC proteins are regulated by a diverse network of proteins, and NPC1 itself is the target of small molecule pharmacology efforts. Nevertheless, it is precisely that tie to diverse and potentially reciprocal processes that complicates targeting NPC-related processes and brings with it risks of off-target effects.
Given the central importance of cholesterol metabolism to AD and the fact that many NPC associated proteins are targets for antipsychotics, it should be no surprise that NPC disease is at the nexus of these diverse processes and highlights the heterogeneity of related diseases such as AD. Finding relationships between AD and neurodevelopmental processes is not unprecedented. Alterations of developmentally programmed gene expression and microchimerism have been evoked in claims that AD is neurodevelopmental in origin [10,14]. Moreover, there are clear links between AD and neurodevelopmental diseases based upon APP expression and metabolism, as in the case of Down's syndrome, in which increases in APP and Aβ due to a gene dosage effect is observed. Conversely excess activity of α-soluble APP is believed to contribute to brain enlargement in autism [149]. Furthermore, the tau pathology which is so central to the link between NPC and AD is also present in numerous other neurodevelopmental disorders, such as hemimegalencephaly, tuberous sclerosis complex and focal cortical dysplasia [150]. NPC disease exemplifies how cholesterol metabolism lies at the nexus of developmental and degenerative processes linking diverse phenotypes to common mechanisms.